Lysosomal targeting peptides and uses thereof

ABSTRACT

The present invention provides further improved compositions and methods for efficient lysosomal targeting based on the GILT technology. Among other things, the present invention provides methods and compositions for targeting lysosomal enzymes to lysosomes using furin-resistant lysosomal targeting peptides. The present invention also provides methods and compositions for targeting lysosomal enzymes to lysosomes using a lysosomal targeting peptide that has reduced or diminished binding affinity for the insulin receptor.

CROSS-REFERENCE TO RELATED APPLICATIONS

This is a continuation of U.S. patent application Ser. No. 12/991,104filed Apr. 25, 2011, which is the National Stage Entry ofPCT/US2009/43207 filed May 7, 2009, which claims the benefit of priorityunder 35 U.S.C. §119(e) of U.S. Provisional Patent Application No.61/051,336 filed May 7, 2008 and U.S. Provisional Patent Application No.61/144,106 filed Jan. 12, 2009, the contents of each of which are herebyincorporated by reference in their entireties.

This application contains, as a separate part of the disclosure, asequence listing in computer-readable form (Filename:40017B_SeqListing.txt; Size: 96,281 bytes; Created: Nov. 5, 2014), whichis incorporated by reference in its entirety.

BACKGROUND

Normally, mammalian lysosomal enzymes are synthesized in the cytosol andtraverse the ER where they are glycosylated with N-linked, high mannosetype carbohydrate. In the golgi, the high mannose carbohydrate ismodified on lysosomal proteins by the addition of mannose-6-phosphate(M6P) which targets these proteins to the lysosome. The M6P-modifiedproteins are delivered to the lysosome via interaction with either oftwo M6P receptors. The most favorable form of modification is when twoM6Ps are added to a high mannose carbohydrate.

More than forty lysosomal storage diseases (LSDs) are caused, directlyor indirectly, by the absence of one or more lysosomal enzymes in thelysosome. Enzyme replacement therapy for LSDs is being actively pursued.Therapy generally requires that LSD proteins be taken up and deliveredto the lysosomes of a variety of cell types in an M6P-dependent fashion.One possible approach involves purifying an LSD protein and modifying itto incorporate a carbohydrate moiety with M6P. This modified materialmay be taken up by the cells more efficiently than unmodified LSDproteins due to interaction with M6P receptors on the cell surface.

The inventors of the present application have previously developed apeptide-based targeting technology that allows more efficient deliveryof therapeutic enzymes to the lysosomes. This proprietary technology istermed Glycosylation Independent Lysosomal Targeting (GILT) because apeptide tag replaces M6P as the moiety targeting the lysosomes. Detailsof the GILT technology are described in U.S. Application PublicationNos. 2003-0082176, 2004-0006008, 2003-0072761, 2005-0281805,2005-0244400, and international publications WO 03/032913, WO 03/032727,WO 02/087510, WO 03/102583, WO 2005/078077, the disclosures of all ofwhich are hereby incorporated by reference.

SUMMARY OF THE INVENTION

The present invention provides further improved compositions and methodsfor efficient lysosomal targeting based on the GILT technology. Amongother things, the present invention provides methods and compositionsfor targeting lysosomal enzymes to lysosomes using furin-resistantlysosomal targeting peptides. The present invention also providesmethods and compositions for targeting lysosomal enzymes to lysosomesusing a lysosomal targeting peptide that has reduced or diminishedbinding affinity for the insulin receptor. The present inventionencompasses unexpected discovery that furin-resistant lysosomaltargeting peptides according to the invention have reduced bindingaffinity for the insulin receptor.

In some embodiments, the present invention provides a furin-resistantIGF-II mutein. In some embodiments, the present invention provides afurin-resistant IGF-II mutein having an amino acid sequence at least 70%identical to mature human IGF-II (SEQ ID NO:1) and a mutation thatabolishes at least one furin protease cleavage site.

In some embodiments, the present invention provides an IGF-II muteincomprising an amino acid sequence at least 70% identical to mature humanIGF-II (SEQ ID NO:1) and a mutation that reduces or diminishes thebinding affinity for the insulin receptor as compared to the wild-typehuman IGF-II.

In some embodiments, the furin-resistant IGF-II mutein has diminishedbinding affinity for the IGF-I receptor relative to the affinity ofnaturally-occurring human IGF-II for the IGF-I receptor.

In some embodiments, the present invention provides a targetedtherapeutic fusion protein containing a lysosomal enzyme; and an IGF-IImutein having an amino acid sequence at least 70% identical to maturehuman IGF-II (SEQ ID NO:1), wherein the IGF-II mutein is resistant tofurin cleavage and binds to the human cation-independentmannose-6-phosphate receptor in a mannose-6-phosphate-independentmanner.

In some embodiments, the present invention provides a targetedtherapeutic fusion protein containing a lysosomal enzyme; and an IGF-IImutein having an amino acid sequence at least 70% identical to maturehuman IGF-II (SEQ ID NO:1), and having diminished binding affinity forthe insulin receptor relative to the affinity of naturally-occurringhuman IGF-II for the insulin receptor; wherein the IGF-II mutein bindsto the human cation-independent mannose-6-phosphate receptor in amannose-6-phosphate-independent manner.

In some embodiments, the present invention provides a targetedtherapeutic fusion protein containing a lysosomal enzyme; and an IGF-IImutein having an amino acid sequence at least 70% identical to maturehuman IGF-II (SEQ ID NO:1), and having diminished binding affinity forthe insulin receptor relative to the affinity of naturally-occurringhuman IGF-II for the insulin receptor; wherein the IGF-II mutein isresistant to furin cleavage and binds to the human cation-independentmannose-6-phosphate receptor in a mannose-6-phosphate-independentmanner.

In some embodiments, an IGF-II mutein suitable for the inventionincludes a mutation within a region corresponding to amino acids 30-40of SEQ ID NO:1. In some embodiments, an IGF-II mutein suitable for theinvention includes a mutation within a region corresponding to aminoacids 34-40 of SEQ ID NO:1 such that the mutation abolishes at least onefurin protease cleavage site. In some embodiments, a suitable mutationis an amino acid substitution, deletion and/or insertion. In someembodiments, the mutation is an amino acid substitution at a positioncorresponding to Arg37 or Arg40 of SEQ ID NO:1. In some embodiments, theamino acid substitution is a Lys or Ala substitution.

In some embodiments, a suitable mutation is a deletion or replacement ofamino acid residues corresponding to positions selected from the groupconsisting of 31-40, 32-40, 33-40, 34-40, 30-39, 31-39, 32-39, 34-37,32-39, 33-39, 34-39, 35-39, 36-39, 37-40, 34-40 of SEQ ID NO:1, andcombinations thereof.

In some embodiments, an IGF-II mutein according to the invention furthercontains a deletion or a replacement of amino acids corresponding topositions 2-7 of SEQ ID NO:1. In some embodiments, an IGF-II muteinaccording to the invention further includes a deletion or a replacementof amino acids corresponding to positions 1-7 of SEQ ID NO:1. In someembodiments, an IGF-II mutein according to the invention furthercontains a deletion or a replacement of amino acids corresponding topositions 62-67 of SEQ ID NO:1. In some embodiments, an IGF-II muteinaccording to the invention further contains an amino acid substitutionat a position corresponding to Tyr27, Leu43, or Ser26 of SEQ ID NO:1. Insome embodiments, an IGF-II mutein according to the invention containsat least an amino acid substitution selected from the group consistingof Tyr27Leu, Leu43Val, Ser26Phe and combinations thereof. In someembodiments, an IGF-II mutein according to the invention contains aminoacids corresponding to positions 48-55 of SEQ ID NO:1. In someembodiments, an IGF-II mutein according to the invention contains atleast three amino acids selected from the group consisting of aminoacids corresponding to positions 8, 48, 49, 50, 54, and 55 of SEQ IDNO:1. In some embodiments, an IGF-11 mutein of the invention contains,at positions corresponding to positions 54 and 55 of SEQ ID NO:1, aminoacids each of which is uncharged or negatively charged at pH 7.4. Insome embodiments, the IGF-II mutein has diminished binding affinity forthe IGF-I receptor relative to the affinity of naturally-occurring humanIGF-II for the IGF-I receptor.

In some embodiments, a lysosomal enzyme suitable for the invention ishuman acid alpha-glucosidase (GAA), or a functional variant thereof. Insome embodiments, a lysosomal enzyme suitable for the invention includesamino acids 70-952 of human GAA.

In some embodiments, a targeted therapeutic fusion protein of theinvention further includes a spacer between the lysosomal enzyme and thefurin-resistant IGF-II mutein. In some embodiments, the spacer containsan amino acid sequence Gly-Ala-Pro.

The present invention also provides nucleic acids encoding the IGF-IImutein or the targeted therapeutic fusion protein as described invarious embodiments above. The present invention further providesvarious cells containing the nucleic acid of the invention.

The present invention provides pharmaceutical compositions suitable fortreating lysosomal storage disease containing a therapeuticallyeffective amount of a targeted therapeutic fusion protein of theinvention. The invention further provides methods of treating lysosomalstorage diseases comprising administering to a subject in need oftreatment a targeted therapeutic fusion protein according to theinvention. In some embodiments, the lysosomal storage disease is PompeDisease. In some embodiments, the lysosomal storage disease is FabryDisease. In some embodiments, the lysosomal storage disease is GaucherDisease.

In another aspect, the present invention provides a method of producinga targeted therapeutic fusion protein including a step of culturingmammalian cells in a cell culture medium, wherein the mammalian cellscarry the nucleic acid of the invention, in particular, as described invarious embodiments herein; and the culturing is performed underconditions that permit expression of the targeted therapeutic fusionprotein.

In yet another aspect, the present invention provides a method ofproducing a targeted therapeutic fusion protein including a step ofculturing furin-deficient cells (e.g., furin-deficient mammalian cells)in a cell culture medium, wherein the furin-deficient cells carry anucleic acid encoding a fusion protein comprising a lysosomal enzyme andan IGF-II mutein having an amino acid sequence at least 70% identical tomature human IGF-II (SEQ ID NO:1), wherein the IGF-II mutein binds tothe human cation-independent mannose-6-phosphate receptor in amannose-6-phosphate-independent manner; and wherein the culturing isperformed under conditions that permit expression of the targetedtherapeutic fusion protein.

Other features, objects, and advantages of the present invention areapparent in the detailed description that follows. It should beunderstood, however, that the detailed description, while indicatingembodiments of the present invention, is given by way of illustrationonly, not limitation. Various changes and modifications within the scopeof the invention will become apparent to those skilled in the art fromthe detailed description.

BRIEF DESCRIPTION OF THE DRAWINGS

The drawings are for illustration purposes only, not for limitation.

FIG. 1 illustrates a map of N-terminus of ZC-701. Two amino acidresidues boxed are sites of cleavage events. The first is the site ofsignal peptide cleavage, the second is the site of a furin cleavage.

FIG. 2 illustrates an exemplary SDS-PAGE analysis of ZC-701 aftertreatment with PNGase F. The lane on the right has been additionallytreated with furin.

FIG. 3 Left: Schematic illustration of exemplary ZC-701 mutants in whichfurin cleavage site is modified. Center: Exemplary SDS-PAGE analysis ofPNGase treated mutants after 3-7 days of cell culture. Right: ExemplarySDS-PAGE analysis of PNGase-treated mutants treated with furin.

FIG. 4 illustrates exemplary competitive IGF-II receptor bindingresults.

FIG. 5 illustrates additional exemplary competitive IGF-II receptorbinding results.

FIG. 6 illustrates exemplary insulin receptor competition assay results.

FIG. 7 illustrates exemplary IGF-I receptor competition assay results.

FIG. 8 illustrates exemplary results of certain insulin receptor bindingassay.

FIG. 9 illustrates exemplary results of certain insulin receptor bindingassay.

FIG. 10 illustrates exemplary analysis of partially purified GILT-taggedGAA from transient transfections. HEK293 cells were transfected withconstructs 1479, 1487 or ZC-701. After harvest, culture supernatantswere partially purified by Hydrophobic Interaction Chromatography (HIC).All samples were treated with PNGase prior to electrophoresis. Leftpanels: SDS-PAGE of partially purified proteins. Purified ZC-701 B12 isshown as a control. Right panels: Immunoblot analysis of the partiallypurified proteins. The indicated primary antibody was used. Bottompanels were additionally treated with exogenous furin. The proteinencoded by construct 1487 is identical in sequence to that encoded byconstruct 1461 (R37A). The protein encoded by construct 1479 isidentical to that encoded by construct 1459 (R37K).

FIG. 11 illustrates exemplary uptake results of exemplary furinresistant GILT-tagged GAA into rat L6 myoblasts. K_(uptakes) for protein1479, 1487, ZC-701, and purified ZC-701 are 4.5 nM, 4.4 nM, 5.0 nM and2.6 nM respectively. The protein encoded by construct 1487 is identicalin sequence to that encoded by construct 1461 in FIG. 3 (R37A). Theprotein encoded by construct 1479 is identical to that encoded byconstruct 1459 in FIG. 3 (R37K).

DEFINITIONS

Amelioration: As used herein, the term “amelioration” is meant theprevention, reduction or palliation of a state, or improvement of thestate of a subject. Amelioration includes, but does not require completerecovery or complete prevention of a disease condition. In someembodiments, amelioration includes reduction of accumulated materialsinside lysosomes of relevant diseases tissues.

Furin-resistant IGF-II mutein: As used herein, the term “furin-resistantIGF-II mutein” refers to an IGF-II-based peptide containing an alteredamino acid sequence that abolishes at least one native furin proteasecleavage site or changes a sequence close or adjacent to a native furinprotease cleavage site such that the furin cleavage is prevented,inhibited, reduced, or slowed down as compared to a wild-type humanIGF-II peptide. As used herein, a furin-resistant IGF-II mutein is alsoreferred to as an IGF-II mutein that is resistant to furin.

Furin protease cleavage site: As used herein, the term “furin proteasecleavage site” (also referred to as “furin cleavage site” or “furincleavage sequence”) refers to the amino acid sequence of a peptide orprotein that serves as a recognition sequence for enzymatic proteasecleavage by furin or furin-like proteases. Typically, a furin proteasecleavage site has a consensus sequence Arg-X-X-Arg (SEQ ID NO: 2), X isany amino acid. The cleavage site is positioned after thecarboxy-terminal arginine (Arg) residue in the sequence. In someembodiments, a furin cleavage site may have a consensus sequenceLys/Arg-X-X-X-Lys/Arg-Arg (SEQ ID NO: 3), X is any amino acid. Thecleavage site is positioned after the carboxy-terminal arginine (Arg)residue in the sequence.

Furin: As used herein, the term “furin” refers to any protease that canrecognize and cleave the furin protease cleavage site as defined herein,including furin or furin-like protease. Furin is also known as pairedbasic amino acid cleaving enzyme (PACE). Furin belongs to thesubtilisin-like proprotein convertase family. The gene encoding furinwas known as FUR (FES Upstream Region).

Furin-deficient cells: As used herein, the term “furin-deficient cells”refers to any cells whose furin protease activity is inhibited, reducedor eliminated. Furin-deficient cells include both mammalian andnon-mammalian cells that do not produce furin or produce reduced amountof furin or defective furin protease.

Glycosylation Independent Lysosomal Targeting: As used herein, the term“glycosylation independent lysosomal targeting” (also referred to as“GILT”) refer to lysosomal targeting that ismannose-6-phosphate-independent.

Human acid alpha-glucosidase: As used herein, the term “human acidalpha-glucosidase” (also referred to as “GAA”) refers to precursorwild-type form of human GAA or a functional variant that is capable ofreducing glycogen levels in mammalian lysosomes or that can rescue orameliorate one or more Pompe disease symptoms.

Improve, increase, or reduce: As used herein, the terms “improve,”“increase” or “reduce,” or grammatical equivalents, indicate values thatare relative to a baseline measurement, such as a measurement in thesame individual prior to initiation of the treatment described herein,or a measurement in a control individual (or multiple controlindividuals) in the absence of the treatment described herein. A“control individual” is an individual afflicted with the same form oflysosomal storage disease (e.g., Pompe disease) as the individual beingtreated, who is about the same age as the individual being treated (toensure that the stages of the disease in the treated individual and thecontrol individual(s) are comparable).

Individual, subject, patient: As used herein, the terms “subject,”“individual” or “patient” refer to a human or a non-human mammaliansubject. The individual (also referred to as “patient” or “subject”)being treated is an individual (fetus, infant, child, adolescent, oradult human) suffering from a lysosomal storage disease, for example,Pompe disease (i.e., either infantile-, juvenile-, or adult-onset Pompedisease) or having the potential to develop a lysosomal storage disease(e.g., Pompe disease).

Lysosomal storage diseases: As used herein, “lysosomal storage diseases”refer to a group of genetic disorders that result from deficiency in atleast one of the enzymes (e.g., acid hydrolases) that are required tobreak macromolecules down to peptides, amino acids, monosaccharides,nucleic acids and fatty acids in lysosomes. As a result, individualssuffering from lysosomal storage diseases have accumulated materials inlysosomes. Exemplary lysosomal storage diseases are listed in Table 1.

Lysosomal enzyme: As used herein, the term “lysosomal enzyme” refers toany enzyme that is capable of reducing accumulated materials inmammalian lysosomes or that can rescue or ameliorate one or morelysosomal storage disease symptoms. Lysosomal enzymes suitable for theinvention include both wild-type or modified lysosomal enzymes and canbe produced using recombinant and synthetic methods or purified fromnature sources. Exemplary lysosomal enzymes are listed in Table 1.

Spacer: As used herein, the term “spacer” (also referred to as “linker”)refers to a peptide sequence between two protein moieties in a fusionprotein. A spacer is generally designed to be flexible or to interpose astructure, such as an alpha-helix, between the two protein moieties. Aspacer can be relatively short, such as the sequence Gly-Ala-Pro (SEQ IDNO: 4) or Gly-Gly-Gly-Gly-Gly-Pro (SEQ ID NO: 5), or can be longer, suchas, for example, 10-25 amino acids in length.

Therapeutically effective amount: As used herein, the term“therapeutically effective amount” refers to an amount of a targetedtherapeutic fusion protein which confers a therapeutic effect on thetreated subject, at a reasonable benefit/risk ratio applicable to anymedical treatment. The therapeutic effect may be objective (i.e.,measurable by some test or marker) or subjective (i.e., subject gives anindication of or feels an effect). In particular, the “therapeuticallyeffective amount” refers to an amount of a therapeutic fusion protein orcomposition effective to treat, ameliorate, or prevent a desired diseaseor condition, or to exhibit a detectable therapeutic or preventativeeffect, such as by ameliorating symptoms associated with the disease,preventing or delaying the onset of the disease, and/or also lesseningthe severity or frequency of symptoms of the disease. A therapeuticallyeffective amount is commonly administered in a dosing regimen that maycomprise multiple unit doses. For any particular therapeutic fusionprotein, a therapeutically effective amount (and/or an appropriate unitdose within an effective dosing regimen) may vary, for example,depending on route of administration, on combination with otherpharmaceutical agents. Also, the specific therapeutically effectiveamount (and/or unit dose) for any particular patient may depend upon avariety of factors including the disorder being treated and the severityof the disorder; the activity of the specific pharmaceutical agentemployed; the specific composition employed; the age, body weight,general health, sex and diet of the patient; the time of administration,route of administration, and/or rate of excretion or metabolism of thespecific fusion protein employed; the duration of the treatment; andlike factors as is well known in the medical arts.

Treatment: As used herein, the term “treatment” (also “treat” or“treating”) refers to any administration of a therapeutic fusion proteinthat partially or completely alleviates, ameliorates, relieves,inhibits, delays onset of, reduces severity of and/or reduces incidenceof one or more symptoms or features of a particular disease, disorder,and/or condition. Such treatment may be of a subject who does notexhibit signs of the relevant disease, disorder and/or condition and/orof a subject who exhibits only early signs of the disease, disorder,and/or condition. Alternatively or additionally, such treatment may beof a subject who exhibits one or more established signs of the relevantdisease, disorder and/or condition. For example, treatment can refer toimprovement of cardiac status (e.g., increase of end-diastolic and/orend-systolic volumes, or reduction, amelioration or prevention of theprogressive cardiomyopathy that is typically found in Pompe disease) orof pulmonary function (e.g., increase in crying vital capacity overbaseline capacity, and/or normalization of oxygen desaturation duringcrying); improvement in neurodevelopment and/or motor skills (e.g.,increase in AIMS score); reduction of glycogen levels in tissue of theindividual affected by the disease; or any combination of these effects.In some embodiments, treatment includes improvement of glycogenclearance, particularly in reduction or prevention of Pompedisease-associated cardiomyopathy.

As used in this application, the terms “about” and “approximately” areused as equivalents. Any numerals used in this application with orwithout about/approximately are meant to cover any normal fluctuationsappreciated by one of ordinary skill in the relevant art.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides improved methods and compositions fortargeting lysosomal enzymes based on the glycosylation-independentlysosomal targeting (GILT) technology. Among other things, the presentinvention provides IGF-II muteins that are resistant to furin and/or hasreduced or diminished binding affinity for the insulin receptor andtargeted therapeutic fusion proteins containing an IGF-II mutein of theinvention. The present invention also provides methods of making andusing the same.

Various aspects of the invention are described in detail in thefollowing sections. The use of sections is not meant to limit theinvention. Each section can apply to any aspect of the invention. Inthis application, the use of “or” means “and/or” unless statedotherwise.

Lysosomal Enzymes

A lysosomal enzyme suitable for the invention includes any enzyme thatis capable of reducing accumulated materials in mammalian lysosomes orthat can rescue or ameliorate one or more lysosomal storage diseasesymptoms. Suitable lysosomal enzymes include both wild-type or modifiedlysosomal enzymes and can be produced using recombinant or syntheticmethods or purified from nature sources. Exemplary lysosomal enzymes arelisted in Table 1.

TABLE 1 Lysosomal Storage Diseases and associated enzyme defects DiseaseName Enzyme Defect Substance Stored A. Glycogenosis Disorders PompeDisease Acid-a1, 4- Glycogen α1-4 linked Glucosidase Oligosaccharides B.Glycolipidosis Disorders GM1 Gangliodsidosis β-Galactosidase GM₁Gangliosides Tay-Sachs Disease β-Hexosaminidase A GM₂ Ganglioside GM2Gangliosidosis: GM₂ Activator GM₂ Ganglioside AB Variant ProteinSandhoff Disease β-Hexosaminidase GM₂ Ganglioside A & B Fabry Diseaseα-Galactosidase A Globosides Gaucher Disease GlucocerebrosidaseGlucosylceramide Metachromatic Arylsulfatase A SulphatidesLeukodystrophy Krabbe Disease Galactosylceramidase GalactocerebrosideNiemann-Pick, Types Acid Sphingomyelin A and B SphingomyelinaseNiemann-Pick, Type C Cholesterol Sphingomyelin Esterification DefectNiemann-Pick, Type D Unknown Sphingomyelin Farber Disease AcidCeramidase Ceramide Wolman Disease Acid Lipase Cholesteryl Esters C.Mucopolysaccharide Disorders Hurler Syndrome α-L-Iduronidase Heparan &(MPS IH) Dermatan Sulfates Scheie Syndrome α-L-Iduronidase Heparan &(MPS IS) Dermatan, Sulfates Hurler-Scheie α-L-Iduronidase Heparan & (MPSIH/S) Dermatan Sulfates Hunter Syndrome Iduronate Sulfatase Heparan &(MPS II) Dermatan Sulfates Sanfilippo A Heparan N-Sulfatase HeparanSulfate (MPS IIIA) Sanfilippo B α-N- Heparan Sulfate (MPS IIIB)Acetylglucosaminidase Sanfilippo C Acetyl-CoA- Heparan Sulfate (MPSIIIC) Glucosaminide Acetyltransferase Sanfilippo D N-Acetylglucosamine-Heparan Sulfate (MPS IIID) 6-Sulfatase Morquio A Galactosamine-6-Keratan Sulfate (MPS IVA) Sulfatase Morquio B β-Galactosidase KeratanSulfate (MPS IVB) Maroteaux-Lamy Arylsulfatase B Dermatan Sulfate (MPSVI) Sly Syndrome β-Glucuronidase (MPS VII) D.Oligosaccharide/Glycoprotein Disorders α-Mannosidosis α-MannosidaseMannose/ Oligosaccharides β-Mannosidosis β-Mannosidase Mannose/Oligosaccharides Fucosidosis α-L-Fucosidase Fucosyl OligosaccharidesAspartylglucosaminuria N-Aspartyl-β- Aspartylglucosamine GlucosaminidaseAsparagines Sialidosis α-Neuraminidase Sialyloligosaccharides(Mucolipidosis I) Galactosialidosis Lysosomal ProtectiveSialyloligosaccharides (Goldberg Syndrome) Protein Deficiency SchindlerDisease α-N-Acetyl- Galactosaminidase E. Lysosomal Enzyme TransportDisorders Mucolipidosis II N-Acetylglucosamine- Heparan Sulfate (I-CellDisease) 1-Phosphotransferase Mucolipidosis III Same as ML II(Pseudo-Hurler Polydystrophy) F. Lysosomal Membrane Transport DisordersCystinosis Cystine Transport Free Cystine Protein Salla Disease SialicAcid Transport Free Sialic Acid and Protein Glucuronic Acid InfantileSialic Acid Sialic Acid Transport Free Sialic Acid and Storage DiseaseProtein Glucuronic Acid G. Other Batten Disease Unknown Lipofuscins(Juvenile Neuronal Ceroid Lipofuscinosis) Infantile NeuronalPalmitoyl-Protein Lipofuscins Ceroid Lipofuscinosis ThioesteraseMucolipidosis IV Unknown Gangliosides & Hyaluronic Acid ProsaposinSaposins A, B, C or D

In some embodiments, a lysosomal enzyme suitable for the inventionincludes a polypeptide sequence having 50-100%, including 50, 55, 60,65, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86,87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 and 100%, sequenceidentity to the naturally-occurring polynucleotide sequence of a humanenzyme shown in Tables 1, while still encoding a protein that is capableof reducing accumulated materials in mammalian lysosomes or that canrescue or ameliorate one or more lysosomal storage disease symptoms.

“Percent (%) amino acid sequence identity” with respect to the lysosomalenzyme sequences is defined as the percentage of amino acid residues ina candidate sequence that are identical with the amino acid residues inthe naturally-occurring human enzyme sequence, after aligning thesequences and introducing gaps, if necessary, to achieve the maximumpercent sequence identity, and not considering any conservativesubstitutions as part of the sequence identity. Alignment for purposesof determining percent amino acid sequence identity can be achieved invarious ways that are within the skill in the art, for instance, usingpublicly available computer software such as BLAST, ALIGN or Megalign(DNASTAR) software. Those skilled in the art can determine appropriateparameters for measuring alignment, including any algorithms needed toachieve maximal alignment over the full length of the sequences beingcompared. Preferably, the WU-BLAST-2 software is used to determine aminoacid sequence identity (Altschul et al., Methods in Enzymology 266,460-480 (1996); http://blast.wust1/edu/blast/README.html). WU-BLAST-2uses several search parameters, most of which are set to the defaultvalues. The adjustable parameters are set with the following values:overlap span=1, overlap fraction=0.125, world threshold (T)=11. HSPscore (S) and HSP S2 parameters are dynamic values and are establishedby the program itself, depending upon the composition of the particularsequence, however, the minimum values may be adjusted and are set asindicated above.

Pompe Disease

One exemplary lysosomal storage disease is Pompe disease. Pompe diseaseis a rare genetic disorder caused by a deficiency in the enzyme acidalpha-glucosidase (GAA), which is needed to break down glycogen, astored form of sugar used for energy. Pompe disease is also known asglycogen storage disease type II, GSD II, type II glycogen storagedisease, glycogenosis type II, acid maltase deficiency,alpha-1,4-glucosidase deficiency, cardiomegalia glycogenic diffusa, andcardiac form of generalized glycogenosis. The build-up of glycogencauses progressive muscle weakness (myopathy) throughout the body andaffects various body tissues, particularly in the heart, skeletalmuscles, liver, respiratory and nervous system.

The presenting clinical manifestations of Pompe disease can vary widelydepending on the age of disease onset and residual GAA activity.Residual GAA activity correlates with both the amount and tissuedistribution of glycogen accumulation as well as the severity of thedisease. Infantile-onset Pompe disease (less than 1% of normal GAAactivity) is the most severe form and is characterized by hypotonia,generalized muscle weakness, and hypertrophic cardiomyopathy, andmassive glycogen accumulation in cardiac and other muscle tissues. Deathusually occurs within one year of birth due to cardiorespiratoryfailure. Hirschhorn et al. (2001) “Glycogen Storage Disease Type II:Acid Alpha-glucosidase (Acid Maltase) Deficiency,” in Scriver et al.,eds., The Metabolic and Molecular Basis of Inherited Disease, 8th Ed.,New York: McGraw-Hill, 3389-3420. Juvenile-onset (1-10% of normal GAAactivity) and adult-onset (10-40% of normal GAA activity) Pompe diseaseare more clinically heterogeneous, with greater variation in age ofonset, clinical presentation, and disease progression. Juvenile- andadult-onset Pompe disease are generally characterized by lack of severecardiac involvement, later age of onset, and slower disease progression,but eventual respiratory or limb muscle involvement results insignificant morbidity and mortality. While life expectancy can vary,death generally occurs due to respiratory failure. Hirschhorn et al.(2001) “Glycogen Storage Disease Type II: Acid Alpha-glucosidase (AcidMaltase) Deficiency,” in Scriver et al., eds., The Metabolic andMolecular Basis of Inherited Disease, 8th Ed., New York: McGraw-Hill,3389-3420.

A GAA enzyme suitable for treating Pompe disease includes a wild-typehuman GAA, or a fragment or sequence variant thereof which retains theability to cleave a 1-4 linkages in linear oligosaccharides.

Enzyme Replacement Therapy

Enzyme replacement therapy (ERT) is a therapeutic strategy to correct anenzyme deficiency by infusing the missing enzyme into the bloodstream.As the blood perfuses patient tissues, enzyme is taken up by cells andtransported to the lysosome, where the enzyme acts to eliminate materialthat has accumulated in the lysosomes due to the enzyme deficiency. Forlysosomal enzyme replacement therapy to be effective, the therapeuticenzyme must be delivered to lysosomes in the appropriate cells intissues where the storage defect is manifest. Conventional lysosomalenzyme replacement therapeutics are delivered using carbohydratesnaturally attached to the protein to engage specific receptors on thesurface of the target cells. One receptor, the cation-independent M6Preceptor (CI-MPR), is particularly useful for targeting replacementlysosomal enzymes because the CI-MPR is present on the surface of mostcell types.

The terms “cation-independent mannose-6-phosphate receptor (CI-MPR),”“M6P/IGF-II receptor,” “CI-MPR/IGF-II receptor,” “IGF-II receptor” or“IGF2 Receptor,” or abbreviations thereof, are used interchangeablyherein, referring to the cellular receptor which binds both M6P andIGF-II.

Glycosylation Independent Lysosomal Targeting

We have developed a Glycosylation Independent Lysosomal Targeting (GILT)technology to target therapeutic enzymes to lysosomes. Specifically, theGILT technology uses a peptide tag instead of M6P to engage the CI-MPRfor lysosomal targeting. Typically, a GILT tag is a protein, peptide, orother moiety that binds the CI-MPR in a mannose-6-phosphate-independentmanner. Advantageously, this technology mimics the normal biologicalmechanism for uptake of lysosomal enzymes, yet does so in a mannerindependent of mannose-6-phosphate.

A preferred GILT tag is derived from human insulin-like growth factor II(IGF-II). Human IGF-II is a high affinity ligand for the CI-MPR, whichis also referred to as IGF-II receptor. Binding of GILT-taggedtherapeutic enzymes to the M6P/IGF-II receptor targets the protein tothe lysosome via the endocytic pathway. This method has numerousadvantages over methods involving glycosylation including simplicity andcost effectiveness, because once the protein is isolated, no furthermodifications need be made.

Detailed description of the GILT technology and GILT tag can be found inU.S. Publication Nos. 20030082176, 20040006008, 20040005309, and20050281805, the teachings of all of which are hereby incorporated byreferences in their entireties.

Furin-Resistant GILT Tag

During the course of development of GILT-tagged lysosomal enzymes fortreating lysosomal storage disease, it has become apparent that theIGF-II derived GILT tag may be subjected to proteolytic cleavage byfurin during production in mammalian cells (see the examples section).Furin protease typically recognizes and cleaves a cleavage site having aconsensus sequence Arg-X-X-Arg (SEQ ID NO: 2), X is any amino acid. Thecleavage site is positioned after the carboxy-terminal arginine (Arg)residue in the sequence. In some embodiments, a furin cleavage site hasa consensus sequence Lys/Arg-X-X-X-Lys/Arg-Arg (SEQ ID NO: 3), X is anyamino acid. The cleavage site is positioned after the carboxy-terminalarginine (Arg) residue in the sequence. As used herein, the term “furin”refers to any protease that can recognize and cleave the furin proteasecleavage site as defined herein, including furin or furin-like protease.Furin is also known as paired basic amino acid cleaving enzyme (PACE).Furin belongs to the subtilisin-like proprotein convertase family thatincludes PC3, a protease responsible for maturation of proinsulin inpancreatic islet cells. The gene encoding furin was known as FUR (FESUpstream Region).

The mature human IGF-II peptide sequence is shown below.

(SEQ ID NO: 1)                                     ↓  ↓AYRPSETLCGGELVDTLQFVCGDRGFYFSRPAS RVSRRSR GIVEECCFRS CDLALLETYCATPAKSE

As can be seen, the mature human IGF-II contains two potentialoverlapping furin cleavage sites between residues 34-40 (bolded andunderlined). Arrows point to two potential furin cleavage positions.

We have developed modified GILT tags that are resistant to cleavage byfurin and still retain ability to bind to the CI-MPR in amannose-6-phosphate-independent manner. Specifically, furin-resistantGILT tags can be designed by mutating the amino acid sequence at one ormore furin cleavage sites such that the mutation abolishes at least onefurin cleavage site. Thus, in some embodiments, a furin-resistant GILTtag is a furin-resistant IGF-II mutein containing a mutation thatabolishes at least one furin protease cleavage site or changes asequence adjacent to the furin protease cleavage site such that thefurin cleavage is prevented, inhibited, reduced or slowed down ascompared to a wild-type IGF-II peptide (e.g., wild-type human matureIGF-II). Typically, a suitable mutation does not impact the ability ofthe furin-resistant GILT tag to bind to the human cation-independentmannose-6-phosphate receptor. In particular, a furin-resistant IGF-IImutein suitable for the invention binds to the human cation-independentmannose-6-phosphate receptor in a mannose-6-phosphate-independent mannerwith a dissociation constant of 10⁻⁷ M or less (e.g., 10⁻⁸, 10⁻⁹, 10⁻¹⁰,10⁻¹¹, or less) at pH 7.4. In some embodiments, a furin-resistant IGF-IImutein contains a mutation within a region corresponding to amino acids30-40 (e.g., 31-40, 32-40, 33-40, 34-40, 30-39, 31-39, 32-39, 34-37,32-39, 33-39, 34-39, 35-39, 36-39, 37-40, 34-40) of SEQ ID NO:1. In someembodiments, a suitable mutation abolishes at least one furin proteasecleavage site. A mutation can be amino acid substitutions, deletions,insertions. For example, any one amino acid within the regioncorresponding to residues 30-40 (e.g., 31-40, 32-40, 33-40, 34-40,30-39, 31-39, 32-39, 34-37, 32-39, 33-39, 34-39, 35-39, 36-39, 37-40,34-40) of SEQ ID NO:1 can be substituted with any other amino acid ordeleted. For example, substitutions at position 34 may affect furinrecognition of the first cleavage site. Insertion of one or moreadditional amino acids within each recognition site may abolish one orboth furin cleavage sites. Deletion of one or more of the residues inthe degenerate positions may also abolish both furin cleavage sites.

In some embodiments, a furin-resistant IGF-II mutein contains amino acidsubstitutions at positions corresponding to Arg37 or Arg40 of SEQ IDNO:1. In some embodiments, a furin-resistant IGF-II mutein contains aLys or Ala substitution at positions Arg37 or Arg40. Other substitutionsare possible, including combinations of Lys and/or Ala mutations at bothpositions 37 and 40, or substitutions of amino acids other than Lys orAla.

In some embodiments, the furin-resistant IGF-II mutein suitable for theinvention may contain additional mutations. For example, up to 30% ormore of the residues of SEQ ID NO:1 may be changed (e.g., up to 1%, 2%,3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%,19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30% or moreresidues may be changed). Thus, a furin-resistant IGF-II mutein suitablefor the invention may have an amino acid sequence at least 70%,including at least 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82,83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99%,identical to SEQ ID NO:1.

In some embodiments, a furin-resistant IGF-II mutein suitable for theinvention is targeted specifically to the CI-MPR. Particularly usefulare mutations in the IGF-II polypeptide that result in a protein thatbinds the CI-MPR with high affinity (e.g., with a dissociation constantof 10⁻⁷M or less at pH 7.4) while binding other receptors known to bebound by IGF-II with reduced affinity relative to native IGF-II. Forexample, a furin-resistant IGF-II mutein suitable for the invention canbe modified to have diminished binding affinity for the IGF-I receptorrelative to the affinity of naturally-occurring human IGF-II for theIGF-I receptor. For example, substitution of IGF-II residues Tyr 27 withLeu, Leu 43 with Val or Ser 26 with Phe diminishes the affinity ofIGF-II for the IGF-I receptor by 94-, 56-, and 4-fold respectively(Torres et al. (1995) J. Mol. Biol. 248(2):385-401). Deletion ofresidues 1-7 of human IGF-II resulted in a 30-fold decrease in affinityfor the human IGF-I receptor and a concomitant 12 fold increase inaffinity for the rat IGF-II receptor (Hashimoto et al. (1995) J. Biol.Chem. 270(30):18013-8). The NMR structure of IGF-II shows that Thr 7 islocated near residues 48 Phe and 50 Ser as well as near the 9 Cys-47 Cysdisulfide bridge. It is thought that interaction of Thr 7 with theseresidues can stabilize the flexible N-terminal hexapeptide required forIGF-I receptor binding (Terasawa et al. (1994) EMBO J. 13(23)5590-7). Atthe same time this interaction can modulate binding to the IGF-IIreceptor. Truncation of the C-terminus of IGF-II (residues 62-67) alsoappear to lower the affinity of IGF-II for the IGF-I receptor by 5 fold(Roth et al. (1991) Biochem. Biophys. Res. Commun. 181(2):907-14).

The binding surfaces for the IGF-I and cation-independent M6P receptorsare on separate faces of IGF-II. Based on structural and mutationaldata, functional cation-independent M6P binding domains can beconstructed that are substantially smaller than human IGF-II. Forexample, the amino terminal amino acids (e.g., 1-7 or 2-7) and/or thecarboxy terminal residues 62-67 can be deleted or replaced.Additionally, amino acids 29-40 can likely be eliminated or replacedwithout altering the folding of the remainder of the polypeptide orbinding to the cation-independent M6P receptor. Thus, a targeting moietyincluding amino acids 8-28 and 41-61 can be constructed. These stretchesof amino acids could perhaps be joined directly or separated by alinker. Alternatively, amino acids 8-28 and 41-61 can be provided onseparate polypeptide chains. Comparable domains of insulin, which ishomologous to IGF-II and has a tertiary structure closely related to thestructure of IGF-II, have sufficient structural information to permitproper refolding into the appropriate tertiary structure, even whenpresent in separate polypeptide chains (Wang et al. (1991) TrendsBiochem. Sci. 279-281). Thus, for example, amino acids 8-28, or aconservative substitution variant thereof, could be fused to a lysosomalenzyme; the resulting fusion protein could be admixed with amino acids41-61, or a conservative substitution variant thereof, and administeredto a patient.

IGF-II can also be modified to minimize binding to serum TGF-bindingproteins (Baxter (2000) Am. J. Physiol Endocrinol Metab. 278(6):967-76)to avoid sequestration of IGF-II/GILT constructs. A number of studieshave localized residues in IGF-II necessary for binding to IGF-bindingproteins. Constructs with mutations at these residues can be screenedfor retention of high affinity binding to the M6P/IGF-II receptor andfor reduced affinity for IGF-binding proteins. For example, replacingPhe 26 of IGF-II with Ser is reported to reduce affinity of IGF-II forIGFBP-1 and -6 with no effect on binding to the M6P/IGF-II receptor(Bach et al. (1993) J. Biol. Chem. 268(13):9246-54). Othersubstitutions, such as Lys for Glu 9, can also be advantageous. Theanalogous mutations, separately or in combination, in a region of IGF-Ithat is highly conserved with IGF-II result in large decreases in IGF-BPbinding (Magee et al. (1999) Biochemistry 38(48):15863-70).

An alternate approach is to identify minimal regions of IGF-II that canbind with high affinity to the M6P/IGF-II receptor. The residues thathave been implicated in IGF-II binding to the M6P/IGF-II receptor mostlycluster on one face of IGF-II (Terasawa et al. (1994) EMBO J.13(23):5590-7). Although IGF-II tertiary structure is normallymaintained by three intramolecular disulfide bonds, a peptideincorporating the amino acid sequence on the M6P/IGF-II receptor bindingsurface of IGF-II can be designed to fold properly and have bindingactivity. Such a minimal binding peptide is a highly preferred lysosomaltargeting domain. For example, a preferred lysosomal targeting domain isamino acids 8-67 of human IGF-II. Designed peptides, based on the regionaround amino acids 48-55, which bind to the M6P/IGF-II receptor, arealso desirable lysosomal targeting domains. Alternatively, a randomlibrary of peptides can be screened for the ability to bind theM6P/IGF-II receptor either via a yeast two hybrid assay, or via a phagedisplay type assay.

Binding Affinity for the Insulin Receptor

The inventors of the present application discovered unexpectedly thatmany furin-resistant IGF-II muteins described herein have reduced ordiminished binding affinity for the insulin receptor. Thus, in someembodiments, a peptide tag suitable for the invention has reduced ordiminished binding affinity for the insulin receptor relative to theaffinity of naturally-occurring human IGF-II for the insulin receptor.In some embodiments, peptide tags with reduced or diminished bindingaffinity for the insulin receptor suitable for the invention includepeptide tags having a binding affinity for the insulin receptor that ismore than 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold,8-fold, 9-fold, 10-fold, 12-fold, 14-fold, 16-fold, 18-fold, 20-fold,50-fold, 100-fold less than that of the wild-type mature human IGF-II.The binding affinity for the insulin receptor can be measured usingvarious in vitro and in vivo assays known in the art. Exemplary bindingassays are described in the Examples section.

Mutagenesis

IGF-II muteins can be prepared by introducing appropriate nucleotidechanges into the IGF-II DNA, or by synthesis of the desired IGF-IIpolypeptide. Variations in the IGF-II sequence can be made, for example,using any of the techniques and guidelines for conservative andnon-conservative mutations set forth, for instance, in U.S. Pat. No.5,364,934. Variations may be a substitution, deletion or insertion ofone or more codons encoding IGF-II that results in a change in the aminoacid sequence of IGF-II as compared with a naturally-occurring sequenceof mature human IGF-II. Amino acid substitutions can be the result ofreplacing one amino acid with another amino acid having similarstructural and/or chemical properties, such as the replacement of aleucine with a serine, i.e., conservative amino acid replacements. Aminoacid substitutions can also be the result of replacing one amino acidwith another amino acid having dis-similar structural and/or chemicalproperties, i.e., non-conservative amino acid replacements. Insertionsor deletions may optionally be in the range of 1 to 5 amino acids. Thevariation allowed may be determined by systematically making insertions,deletions or substitutions of amino acids in the sequence and testingthe resulting variants for activity in the in vivo or in vitro assaysknown in the art (such as binding assays to the CI-MPR or furin cleavageassays).

Scanning amino acid analysis can also be employed to identify one ormore amino acids along a contiguous sequence. Among the preferredscanning amino acids are relatively small, neutral amino acids. Suchamino acids include alanine, glycine, serine, and cysteine. Alanine istypically a preferred scanning amino acid among this group because iteliminates the side-chain beyond the beta-carbon and is less likely toalter the main-chain conformation of the variant. Alanine is alsotypically preferred because it is the most common amino acid. Further,it is frequently found in both buried and exposed positions [Creighton,The Proteins, (W. H. Freeman & Co., N.Y.); Chothia, J. Mol. Biol., 150:1(1976)]. If alanine substitution does not yield adequate amounts ofvariant, an isoteric amino acid can be used.

The variations can be made using methods known in the art such asoligonucleotide-mediated (site-directed) mutagenesis, alanine scanning,and PCR mutagenesis. Site-directed mutagenesis [Carter et al., Nucl.Acids Res., 13:4331 (1986); Zoller et al., Nucl. Acids Res., 10:6487(1987)], cassette mutagenesis [Wells et al., Gene, 34:315 (1985)],restriction selection mutagenesis [Wells et al., Philos. Trans. R. Soc.London SerA, 317:415 (1986)] or other known techniques can be performedon the cloned DNA to produce IGF-II muteins.

Spacer

A furin-resistant GILT tag can be fused to the N-terminus or C-terminusof a polypeptide encoding a lysosomal enzyme. The GILT tag can be fuseddirectly to the lysosomal enzyme polypeptide or can be separated fromthe lysosomal enzyme polypeptide by a linker or a spacer. An amino acidlinker or spacer is generally designed to be flexible or to interpose astructure, such as an alpha-helix, between the two protein moieties. Alinker or spacer can be relatively short, such as the sequenceGly-Ala-Pro (SEQ ID NO: 4) or Gly-Gly-Gly-Gly-Gly-Pro (SEQ ID NO: 5), orcan be longer, such as, for example, 10-25 amino acids in length. Thesite of a fusion junction should be selected with care to promote properfolding and activity of both fusion partners and to prevent prematureseparation of a peptide tag from a GAA polypeptide. In a preferredembodiment, the linker sequence is Gly-Ala-Pro (SEQ ID NO: 4).

Additional constructs of GILT-tagged GAA proteins that can be used inthe methods and compositions of the present invention were described indetail in U.S. Publication No. 20050244400, the entire disclosure ofwhich is incorporated herein by reference.

Cells

Any mammalian cell or cell type susceptible to cell culture, and toexpression of polypeptides, may be utilized in accordance with thepresent invention, such as, for example, human embryonic kidney (HEK)293, Chinese hamster ovary (CHO), monkey kidney (COS), HT1080, C10,HeLa, baby hamster kidney (BHK), 3T3, C127, CV-1, HaK, NS/O, and L-929cells. Non-limiting examples of mammalian cells that may be used inaccordance with the present invention include, but are not limited to,BALB/c mouse myeloma line (NSO/I, ECACC No: 85110503); humanretinoblasts (PER.C6 (CruCell, Leiden, The Netherlands)); monkey kidneyCV1 line transformed by SV40 (COS-7, ATCC CRL 1651); human embryonickidney line (293 or 293 cells subcloned for growth in suspensionculture, Graham et al., J. Gen Virol., 36:59 (1977)); baby hamsterkidney cells (BHK, ATCC CCL 10); Chinese hamster ovary cells+/−DHFR(CHO, Urlaub and Chasin, Proc. Natl. Acad. Sci. USA, 77:4216 (1980));mouse sertoli cells (TM4, Mather, Biol. Reprod., 23:243-251 (1980));monkey kidney cells (CV1 ATCC CCL 70); African green monkey kidney cells(VERO-76, ATCC CRL-1 587); human cervical carcinoma cells (HeLa, ATCCCCL 2); canine kidney cells (MDCK, ATCC CCL 34); buffalo rat liver cells(BRL 3A, ATCC CRL 1442); human lung cells (W138, ATCC CCL 75); humanliver cells (Hep G2, HB 8065); mouse mammary tumor (MMT 060562, ATCCCCL51); TRI cells (Mather et al., Annals N.Y. Acad. Sci., 383:44-68(1982)); MRC 5 cells; FS4 cells; and a human hepatoma line (Hep G2). Insome embodiments, the fusion protein of the present invention isproduced from CHO cell lines.

The fusion protein of the invention can also be expressed in a varietyof non-mammalian host cells such as, for example, insect (e.g., Sf-9,Sf-21, Hi5), plant (e.g., Leguminosa, cereal, or tobacco), yeast (e.g.,S. cerivisae, P. pastoris), prokaryote (e.g., E. Coli, B. subtilis andother Bacillus spp., Pseudomonas spp., Streptomyces spp), or fungus.

In some embodiments, a fusion protein with or without a furin-resistantGILT tag can be produced in furin-deficient cells. As used herein, theterm “furin-deficient cells” refers to any cells whose furin proteaseactivity is inhibited, reduced or eliminated. Furin-deficient cellsinclude both mammalian and non-mammalian cells that do not produce furinor produce reduced amount or defective furin protease. Exemplary furindeficient cells that are known and available to the skilled artisan,including but not limited to FD11 cells (Gordon et al (1997) Infectionand Immunity 65(8):3370 3375), and those mutant cells described inMoebring and Moehring (1983) Infection and Immunity 41(3):998 1009.Alternatively, a furin deficient cell may be obtained by exposing theabove-described mammalian and non-mammalian cells to mutagenesistreatment, e.g., irradiation, ethidium bromide, bromidated uridine(BrdU) and others, preferably chemical mutagenesis, and more preferredethyl methane sulfonate mutagenesis, recovering the cells which survivethe treatment and selecting for those cells which are found to beresistant to the toxicity of Pseudomonas exotoxin A (see Moehring andMoehrin (1983) Infection and Immunity 41(3):998 1009).

Underglycosylation

Targeted therapeutic proteins of the invention can be underglycosylated,that is, one or more carbohydrate structures that would normally bepresent on a naturally-occurring human protein is preferably omitted,removed, modified, or masked. Without wishing to be bound by anytheories, it is contemplated that an underglycosylated protein mayextend the half-life of the protein in a mammal. Underglycosylation canbe achieved in many ways. In some embodiments, the targeted fusionprotein of the invention can be produced using a secretory signalpeptide to facilitate secretion of the fusion protein. For example, thefusion protein can be produced using an IGF-II signal peptide. Ingeneral, the fusion protein produced using an IGF-II signal peptide hasreduced mannose-6-phosphate (M6P) level on the surface of the proteincompared to wild-type enzyme. In some embodiments, a protein may becompletely underglycosylated (as when synthesized in E. coli), partiallyunglycosylated (as when synthesized in a mammalian system afterdisruption of one or more glycosylation sites by site-directedmutagenesis), or may have a non-mammalian glycosylation pattern. Forexample, underglycosylated fusion proteins may be generated bymodifying, substituting or eliminating one or more glycosylation sitesby site-directed mutagenesis. For example, wild-type GAA typically haveseven sites that match the canonical recognition sequence for N-linkedglycosylation, Asn-Xaa-Thr/Ser (SEQ ID NO: 7) (Xaa can be any residueexcept Pro), namely, Asn-140, -233, -390, -470, -652, -882 and -925(Hoefsloot et al., 1988; Martiniuk et al., 1990b). One or more Asn atthe above described positions may be changed or eliminated to generatedunderglycosylated GAA. In some embodiments, Asn may be changed to Gln.

In some embodiments, a therapeutic fusion protein can be deglycosylatedafter synthesis. For example, deglycosylation can be through chemical orenzymatic treatments, and may lead to complete deglycosylation or, ifonly a portion of the carbohydrate structure is removed, partialdeglycosylation.

In some embodiments, glycosylation of a lysosomal enzyme is modified,e.g., by oxidation and reduction, to reduce clearance of the therapeuticprotein from the blood. For example, a lysosomal enzyme can bedeglycosylated by periodate treatment. In particular, treatment withperiodate and a reducing agent such as sodium borohydride is effectiveto modify the carbohydrate structure of most glycoproteins. Periodatetreatment oxidizes vicinal diols, cleaving the carbon-carbon bond andreplacing the hydroxyl groups with aldehyde groups; borohydride reducesthe aldehydes to hydroxyls. For example, at 1 mM concentration,periodate exclusively oxidizes sialic acid groups and at or above 10 mMall available vicinal diols are converted to aldehydes (Hermanson, G. T.1996, Bioconjugate techniques. Academic press). Once formed, aldehydegroups are highly reactive and may form Schiff's base linkages withprimary amino groups in the protein resulting intramolecular linkages.Therefore, aldehyde groups formed ought to be reduced to alcohol groups.A commonly used reducing agent is NaBH₄ and the reaction is best rununder alkaline conditions. Many sugar residues including vicinal diols,therefore, are cleaved by this treatment. Nevertheless, while thistreatment converts cyclic carbohydrates into linear carbohydrates, itdoes not completely remove the carbohydrate, minimizing risks ofexposing potentially protease-sensitive or antigenic polypeptide sites.

Grubb, J. H., et al (Grubb et al, 2008, PNAS 105:2616) report treatmentof human β-glucuronidase with sodium metaperiodate followed by sodiumborohydride reduction. The modified beta-glucuronidase retained 90% ofactivity, but lost both mannose and mannose-6-phosphate dependentreceptor uptake activity. The alkaline pH condition used in thereduction due to sodium borohydride reagent as described by Grubb et alis not suitable for all lysosomal enzymes, many of which are labileunder alkaline conditions.

Therefore, in some embodiments, sodium cyanoborohydride is used asreducing agent. While the rate of reduction of aldehydes bycyanoborohydride is negligible at neutral pH and above, the rate ofreaction becomes rapid at acidic pH (Borch, et al. 1971, JACS 93:2897).For example, regimens using sodium metaperiodate and cyanoborohydride atpH 3.5-4 can be used.

For example, treatment of GAA or alpha galactosidase A, the enzymesdeficient in Pompe and Fabry diseases respectively, with periodate andcyanoborohydride at pH 5.6 resulted in good recovery of enzyme activity.Enzyme was incubated with equal volume mixture containing 20 mM sodiummetaperiodate and 40 mM sodium cyanoborohydride in 0.1 M Na acetate, pH5.6 for 60 min on ice. The unreacted periodate was quenched withglycerol (10% final concentration) for 15 min on ice. The proteins werefinally exchanged into phosphate buffered saline, pH 6.2 bydiafiltration using Amicon centrifugal filter devices. Other reducingreagents for example, dimethylamine borane, may also be useful to reducealdehydes generated by sodium metaperiodate oxidation of glycoproteinssuch as GAA under acidic conditions.

Thus, in some embodiments, the reduction of sodium metaperiodate treatedGAA involves use of sodium cyanoborohydride at acidic pH from pH 3.0 topH 6. Optimal conditions for the chemical modification can be readilydetermined by using two assays: loss of binding to ConA sepharose, anddiminished uptake into J774E macrophage.

For example, the ability of periodate/borohydride modifiedβ-glucuronidase to bind to ConA-sepharose was compared to that ofuntreated β-glucuronidase. The enzymes were incubated with 50 μl ConAbeads in 20 mM Tris-HCl, pH 6.8, 0.5 M NaCl for 15 min at roomtemperature. Beads were centrifuged at maximum speed for 15 sec.Supernatant (flow through) was carefully withdrawn, assayed for GUSactivity and analyzed by SDS/PAGE. When we treated GUS exactly asreported in Grubb et al., 60% ConA binding activity was lost and unboundGUS was present only in the flow through of periodate treated andsubsequently sodium borohydride reduced sample.

Administration of Therapeutic Proteins

In accordance of the invention, a therapeutic protein of the inventionis typically administered to the individual alone, or in compositions ormedicaments comprising the therapeutic protein (e.g., in the manufactureof a medicament for the treatment of the disease), as described herein.The compositions can be formulated with a physiologically acceptablecarrier or excipient to prepare a pharmaceutical composition. Thecarrier and composition can be sterile. The formulation should suit themode of administration.

Suitable pharmaceutically acceptable carriers include but are notlimited to water, salt solutions (e.g., NaCl), saline, buffered saline,alcohols, glycerol, ethanol, gum arabic, vegetable oils, benzylalcohols, polyethylene glycols, gelatin, carbohydrates such as lactose,amylose or starch, sugars such as mannitol, sucrose, or others,dextrose, magnesium stearate, talc, silicic acid, viscous paraffin,perfume oil, fatty acid esters, hydroxymethylcellulose, polyvinylpyrolidone, etc., as well as combinations thereof. The pharmaceuticalpreparations can, if desired, be mixed with auxiliary agents (e.g.,lubricants, preservatives, stabilizers, wetting agents, emulsifiers,salts for influencing osmotic pressure, buffers, coloring, flavoringand/or aromatic substances and the like) which do not deleteriouslyreact with the active compounds or interference with their activity. Ina preferred embodiment, a water-soluble carrier suitable for intravenousadministration is used.

The composition or medicament, if desired, can also contain minoramounts of wetting or emulsifying agents, or pH buffering agents. Thecomposition can be a liquid solution, suspension, emulsion, tablet,pill, capsule, sustained release formulation, or powder. The compositioncan also be formulated as a suppository, with traditional binders andcarriers such as triglycerides. Oral formulation can include standardcarriers such as pharmaceutical grades of mannitol, lactose, starch,magnesium stearate, polyvinyl pyrollidone, sodium saccharine, cellulose,magnesium carbonate, etc.

The composition or medicament can be formulated in accordance with theroutine procedures as a pharmaceutical composition adapted foradministration to human beings. For example, in a preferred embodiment,a composition for intravenous administration typically is a solution insterile isotonic aqueous buffer. Where necessary, the composition mayalso include a solubilizing agent and a local anesthetic to ease pain atthe site of the injection. Generally, the ingredients are suppliedeither separately or mixed together in unit dosage form, for example, asa dry lyophilized powder or water free concentrate in a hermeticallysealed container such as an ampule or sachette indicating the quantityof active agent. Where the composition is to be administered byinfusion, it can be dispensed with an infusion bottle containing sterilepharmaceutical grade water, saline or dextrose/water. Where thecomposition is administered by injection, an ampule of sterile water forinjection or saline can be provided so that the ingredients may be mixedprior to administration.

The therapeutic protein can be formulated as neutral or salt forms.Pharmaceutically acceptable salts include those formed with free aminogroups such as those derived from hydrochloric, phosphoric, acetic,oxalic, tartaric acids, etc., and those formed with free carboxyl groupssuch as those derived from sodium, potassium, ammonium, calcium, ferrichydroxides, isopropylamine, triethylamine, 2-ethylamino ethanol,histidine, procaine, etc.

A therapeutic protein (or a composition or medicament containing atherapeutic protein) is administered by any appropriate route. In apreferred embodiment, a therapeutic protein is administeredintravenously. In other embodiments, a therapeutic protein isadministered by direct administration to a target tissue, such as heartor muscle (e.g., intramuscular), or nervous system (e.g., directinjection into the brain; intraventricularly; intrathecally).Alternatively, a therapeutic protein (or a composition or medicamentcontaining a therapeutic protein) can be administered parenterally,transdermally, or transmucosally (e.g., orally or nasally). More thanone route can be used concurrently, if desired.

A therapeutic protein (or a composition or medicament containing atherapeutic protein) can be administered alone, or in conjunction withother agents, such as antihistamines (e.g., diphenhydramine) orimmunosuppressants or other immunotherapeutic agents which counteractanti-GILT-tagged lysosomal enzyme antibodies. The term, “in conjunctionwith,” indicates that the agent is administered prior to, at about thesame time as, or following the therapeutic protein (or a composition ormedicament containing the therapeutic protein). For example, the agentcan be mixed into a composition containing the therapeutic protein, andthereby administered contemporaneously with the therapeutic protein;alternatively, the agent can be administered contemporaneously, withoutmixing (e.g., by “piggybacking” delivery of the agent on the intravenousline by which the therapeutic protein is also administered, or viceversa). In another example, the agent can be administered separately(e.g., not admixed), but within a short time frame (e.g., within 24hours) of administration of the therapeutic protein.

The therapeutic protein (or composition or medicament containing thetherapeutic protein) is administered in a therapeutically effectiveamount (i.e., a dosage amount that, when administered at regularintervals, is sufficient to treat the disease, such as by amelioratingsymptoms associated with the disease, preventing or delaying the onsetof the disease, and/or also lessening the severity or frequency ofsymptoms of the disease, as described above). The dose which will betherapeutically effective for the treatment of the disease will dependon the nature and extent of the disease's effects, and can be determinedby standard clinical techniques. In addition, in vitro or in vivo assaysmay optionally be employed to help identify optimal dosage ranges usingmethods known in the art. The precise dose to be employed will alsodepend on the route of administration, and the seriousness of thedisease, and should be decided according to the judgment of apractitioner and each patient's circumstances. Effective doses may beextrapolated from dose-response curves derived from in vitro or animalmodel test systems. The therapeutically effective dosage amount can be,for example, about 0.1-1 mg/kg, about 1-5 mg/kg, about 5-20 mg/kg, about20-50 mg/kg, or 20-100 mg/kg. The effective dose for a particularindividual can be varied (e.g., increased or decreased) over time,depending on the needs of the individual. For example, in times ofphysical illness or stress, or if disease symptoms worsen, the dosageamount can be increased.

The therapeutically effective amount of the therapeutic protein (orcomposition or medicament containing the therapeutic protein) isadministered at regular intervals, depending on the nature and extent ofthe disease's effects, and on an ongoing basis. Administration at an“interval,” as used herein, indicates that the therapeutically effectiveamount is administered periodically (as distinguished from a one-timedose). The interval can be determined by standard clinical techniques.In some embodiments, the therapeutic protein is administered bimonthly,monthly, twice monthly, triweekly, biweekly, weekly, twice weekly,thrice weekly, or daily. The administration interval for a singleindividual need not be a fixed interval, but can be varied over time,depending on the needs of the individual. For example, in times ofphysical illness or stress, or if disease symptoms worsen, the intervalbetween doses can be decreased.

As used herein, the term “bimonthly” means administration once per twomonths (i.e., once every two months); the term “monthly” meansadministration once per month; the term “triweekly” means administrationonce per three weeks (i.e., once every three weeks); the term “biweekly”means administration once per two weeks (i.e., once every two weeks);the term “weekly” means administration once per week; and the term“daily” means administration once per day.

The invention additionally pertains to a pharmaceutical compositioncomprising a therapeutic protein, as described herein, in a container(e.g., a vial, bottle, bag for intravenous administration, syringe,etc.) with a label containing instructions for administration of thecomposition for treatment of Pompe disease, such as by the methodsdescribed herein.

The invention will be further and more specifically described by thefollowing examples. Examples, however, are included for illustrationpurposes, not for limitation.

EXAMPLES Example 1 Furin Cleaves an IGF-II Based GILT Tag

ZC-701 has been developed for the treatment of Pompe disease. ZC-701 isa chimeric protein that contains an N-terminal IGF-II based GILT tagfused via a three amino acid spacer to residues 70-952 of humanacid-α-glucosidase (hGAA). Specifically, ZC-701 includes amino acids 1and 8-67 of human IGF-II (i.e., Δ2-7 of mature human IGF-II), the spacersequence Gly-Ala-Pro, and amino acids 70-952 of human GAA. The fulllength amino acid sequence is shown below. The spacer sequence isbolded. The sequence N-terminal to the spacer sequence reflects aminoacids 1 and 8-67 of human IGF-II and the sequence C-terminal to thespacer sequence reflects amino acids 70-952 of human GAA. The twopotential overlapping furin cleavage sites within the IGF-II tagsequence is bolded and underlined. Arrows point to two potential furincleavage positions.

(SEQ ID NO: 8)                                ↓  ↓AALCGGELVDTLQFVCGDRGFYFSRPAS RVSRRSR GIVEECCFRSCDLALLETYCATPAKSEGAPAHPGRPRAVPTQCDVPPNSRFDCAPDKAITQEQCEARGCCYIPAKQGLQGAQMGQPWCFFPPSYPSYKLENLSSSEMGYTATLTRTTPTFFPKDILTLRLDVMMETENRLHFTIKDPANRRYEVPLETPRVHSRAPSPLYSVEFSEEPFGVIVHRQLDGRVLLNTTVAPLFFADQFLQLSTSLPSQYITGLAEHLSPLMLSTSWTRITLWNRDLAPTPGANLYGSHPFYLALEDGGSAHGVFLLNSNAMDVVLQPSPALSWRSTGGILDVYIFLGPEPKSVVQQYLDVVGYPFMPPYWGLGFHLCRWGYSSTAITRQVVENMTRAHFPLDVQWNDLDYMDSRRDFTFNKDGFRDFPAMVQELHQGGRRYMMIVDPAISSSGPAGSYRPYDEGLRRGVFITNETGQPLIGKVWPGSTAFPDFTNPTALAWWEDMVAEFHDQVPFDGMWIDMNEPSNFIRGSEDGCPNNELENPPYVPGVVGGTLQAATICASSHQFLSTHYNLHNLYGLTEAIASHRALVKARGTRPFVISRSTFAGHGRYAGHWTGDVWSSWEQLASSVPEILQFNLLGVPLVGADVCGFLGNTSEELCVRWTQLGAFYPFMRNHNSLLSLPQEPYSFSEPAQQAMRKALTLRYALLPHLYTLFHQAHVAGETVARPLFLEFPKDSSTWTVDHQLLWGEALLITPVLQAGKAEVTGYFPLGTWYDLQTVPIEALGSLPPPPAAPREPAIHSEGQWVTLPAPLDTINVHLRAGYITPLQGPGLTTTESRQQPMALAVALTKGGEARGELFWDDGESLEVLERGAYTQVIFLARNNTIVNELVRVTSEGAGLQLQKVTVLGVATAPQQVLSNGVPVSNFTYSPDTKVLDICVSLLMGEQFLVSWC

During the course of development of ZC-701, it has become apparent thatthe IGF-II derived GILT tag on a fraction of the ZC-701 molecules issubjected to proteolytic cleavage by furin during production in CHOcells. N-terminal analysis of ZC-701 batch 10-2-F45-54 revealed thepresence of two n-terminal sequences. One conformed to the predictedn-terminus of ZC-701 indicating the presence of the predicted ZC-701protein. The other n-terminal sequence aligned with sequence within thetag portion of ZC-701 indicating the presence of a derivative of ZC-701consistent with an endoproteolytic cleavage at amino acid residue 34 ofZC-701. Based on the estimated molar ratios of the two n-termini, thisbatch of ZC-701 was found to have about a 1:1 ratio of intact andcleaved species.

Upon receipt of this result, each of the other batches of ZC-701 weresubjected to n-terminal sequencing. All of the batches displayed thesame two n-termini with the cleaved species ranging from 20-50% of thetotal compound. One batch, previously shown to have low uptake activity,displayed a set of n-termini indicative of additional proteolysis. Weconcluded that the proteolytic event responsible for the second speciesin all of our batches of ZC-701 was perpetrated by furin or a furin-likeprotease.

FIG. 1 shows a map of the amino terminus of ZC-701. The two amino acidboxed residues are the sites of n-termini mapped in all of the ZC-701batches. The first of the N-termini is the site of signal peptidecleavage, which yields the predicted n-terminus of ZC-701. The secondboxed residue is the site of an undesired proteolytic cleavage event.The amino acid sequence proximal to the cleavage site is Arg-Arg-Ser-Arg(SEQ ID NO: 9). This matches the canonical cleavage site of a proteasepresent in CHO cells called furin, which cleaves after Arg-X-X-Arg (SEQID NO: 10). Furin is a member of a family of prohormone convertases thatincludes PC3, a protease responsible for maturation of proinsulin inpancreatic islet cells. In fact the PC3 cleavage site in proinsulin isconserved and identical to the site at which furin cleaves the IGF-IItag.

The Furin cleaved ZC-701 differs in molecular weight from intact ZC-701by about 3000 daltons, which represents less than a 3% difference inmolecular weight. Due to the heterogeneity of the oligosaccharide in theprotein, the presence of the cleaved ZC-701 was not previously detectedby SDS-PAGE. However, if ZC-701 is first deglycosylated by treatmentwith Peptide N-Glycosidase F (PNGase F), then the cleaved protein can beresolved from the intact ZC-701 by SDS-PAGE.

As shown in FIG. 2, lane 1 of the SDS-PAGE gel shows the electrophoreticpattern of deglycosylated purified ZC-701. Two bands are evident. Theupper band is believed to be intact ZC-701 and the lower band isbelieved to be furin cleaved ZC-701. To prove that the lower band isindeed Furin cleaved ZC-701, same proteins loaded in lane 1 were firsttreated with furin and then loaded in lane 2. As shown in FIG. 2, all ofthe proteins in lane 2 co-migrates with the lower band in lane 1indicating that the lower band is in fact furin cleaved ZC-701.

We have estimated the proportion of ZC-701 that has been cleaved withfurin in a number of batches of ZC-701 by quantification of the bandintensity in SDS-PAGE and by quantification of amino acids released inN-terminal sequencing experiments. As discussed above, the fraction ofcleaved ZC-701 has ranged from 20% to 50% in different batches.

Example 2 Targeted Fusion Proteins Containing a Furin-Resistant IGF-IIBased GILT Tag

We can design around the problem of furin cleavage by altering the aminoacid sequence of IGF-II such that the amino acid alteration abolishes atleast one furin cleavage site. A series of mutant versions of ZC-701were generated and assayed for resistance to cleavage by furin.Exemplary mutant versions of ZC-701 were generated as described below.

ZC-701

The GILTΔ2-7-GAA70-952 cassette below was cloned using the Asp718 andNotI sites of the cassette and vector pCEP4 to producepCEP-GILTΔ2-7-GAA70-952 (Plasmid p701). Restriction sites for cloningare in lowercase bold. The spacer amino acid sequence Gly, Ala, Pro(underlined sequence) separate the GAA gene and GILTΔ2-7 tag (upper casesequence). The spacer and tag are placed upstream of GAA residue Ala70.

(SEQ ID NO: 11) ggtaccagctgctagcaagctaattcacaccaATGGGAATCCCAATGGGGAAGTCGATGCTGGTGCTTCTCACCTTCTTGGCCTTCGCCTCGTGCTGCATTGCTGCTCTGTGCGGCGGGGAGCTGGTGGACACCCTCCAGTTCGTCTGTGGGGACCGCGGCTTCTACTTCAGCAGGCCCGCAAGCCGTGTGAGCCGTCGCAGCCGTGGCATCGTTGAGGAGTGCTGTTTCCGCAGCTGTGACCTGGCCCTCCTGGAGACGTACTGTGCTACCCCCGCCAAGTCCGAGGGCGCGCCGgcacaccccggccgtcccagagcagtgcccacacagtgcgacgtcccccccaacagccgcttcgattgcgcccctgacaaggccatcacccaggaacagtgcgaggcccgcggctgctgctacatccctgcaaagcaggggctgcagggagcccagatggggcagccctggtgcttcttcccacccagctaccccagctacaagctggagaacctgagctcctctgaaatgggctacacggccaccctgacccgtaccacccccaccttcttccccaaggacatcctgaccctgcggctggacgtgatgatggagactgagaaccgcctccacttcacgatcaaagatccagctaacaggcgctacgaggtgcccttggagaccccgcgtgtccacagccgggcaccgtccccactctacagcgtggagttctctgaggagccatcggggtgatcgtgcaccggcagctggacggccgcgtgctgctgaacacgacggtggcgcccctgttctttgcggaccagttccttcagctgtccacctcgctgccctcgcagtatatcacaggcctcgccgagcacctcagtcccctgatgctcagcaccagctggaccaggatcaccctgtggaaccgggaccttgcgcccacgcccggtgcgaacctctacgggtctcaccctttctacctggcgctggaggacggcgggtcggcacacggggtgttcctgctaaacagcaatgccatggatgtggtcctgcagccgagccctgcccttagctggaggtcgacaggtgggatcctggatgtctacatcttcctgggcccagagcccaagagcgtggtgcagcagtacctggacgagtgggatacccgttcatgccgccatactggggcctgggcttccacctgtgccgctggggctactcctccaccgctatcacccgccaggtggtggagaacatgaccagggcccacttccccctggacgtccaatggaacgacctggactacatggactcccggagggacttcacgttcaacaaggatggcttccgggacttcccggccatggtgcaggagctgcaccagggcggccggcgctacatgatgatcgtggatcctgccatcagcagctcgggccctgccgggagctacaggccctacgacgagggtctgcggaggggggttttcatcaccaacgagaccggccagccgctgattgggaaggtatggcccgggtccactgccttccccgacttcaccaaccccacagccctggcctggtgggaggacatggtggctgagttccatgaccaggtgcccttcgacggcatgtggattgacatgaacgagccttccaacttcatcaggggctctgaggacggctgccccaacaatgagctggagaacccaccctacgtgcctggggtggttggggggaccctccaggcggcaaccatctgtgcctccagccaccagtactctccacacactacaacctgcacaacctctacggcctgaccgaagccatcgcctcccacagggcgctggtgaaggctcgggggacacgcccatttgtgatctcccgctcgacctttgctggccacggccgatacgccggccactggacgggggacgtgtggagctcctgggagcagctcgcctcctccgtgccagaaatcctgcagtttaacctgctgggggtgcctctggtcggggccgacgtctgcggcttcctgggcaacacctcagaggagctgtgtgtgcgctggacccagctgggggccttctaccccttcatgcggaaccacaacagcctgctcagtctgccccaggagccgtacagatcagcgagccggcccagcaggccatgaggaaggccctcaccctgcgctacgcactcctcccccacctctacacgctgttccaccaggcccacgtcgcgggggagaccgtggcccggcccctcttcctggagttccccaaggactctagcacctggactgtggaccaccagctcctgtggggggaggccctgctcatcaccccagtgctccaggccgggaaggccgaagtgactggctacttccccttgggcacatggtacgacctgcagacggtgccaatagaggcccttggcagcctcccacccccacctgcagctccccgtgagccagccatccacagcgaggggcagtgggtgacgctgccggcccccctggacaccatcaacgtccacctccgggctgggtacatcatccccctgcagggccctggcctcacaaccacagagtcccgccagcagcccatggccctggctgtggccctgaccaagggtggagaggcccgaggggagctgttctgggacgatggagagagcctggaagtgctggagcgaggggcctacacacaggtcatcttcctggccaggaataacacgatcgtgaatgagctggtacgtgtgaccagtgagggagctggcctgcagctgcagaaggtgactgtcctgggcgtggccacggcgccccagcaggtcctctccaacggtgtccctgtctccaacttcacctacagccccgacaccaaggtcctggacatctgtgtctcgctgttgatgggagagcagtttctcgtcagctggtgttagtctaga gcttgctagcggccgcConstruct 1459

The GILTΔ2-7/K37-GAA70-952 cassette below was cloned using the Asp718and NotI sites of the cassette and vector pCEP4 to producepCEP-GILTΔ2-7/K37-GAA70-952 (Plasmid p1459). Restriction sites forcloning are in lowercase bold. The spacer amino acid sequence Gly, Ala,Pro (underlined sequence) separate the GAA gene and GILTΔ2-7/K37 tag(upper case sequence). The spacer and tag are placed upstream of GAAresidue Ala70. The GILTΔ2-7/K37 cassette contains an Arg to Lyssubstitution at amino acid 37 of the human IGF-II sequence (uppercasebold).

(SEQ ID NO: 12) ggtaccagctgctagcaagctaattcacaccaATGGGAATCCCAATGGGGAAGTCGATGCTGGTGCTTCTCACCTTCTTGGCCTTCGCCTCGTGCTGCATTGCTGCTCTGTGCGGCGGGGAGCTGGTGGACACCCTCCAGTTCGTCTGTGGGGACCGCGGCTTCTACTTCAGCAGGCCCGCAAGCCGTGTGAGCAAGCGCAGCCGTGGCATCGTTGAGGAGTGCTGTTTCCGCAGCTGTGACCTGGCCCTCCTGGAGACGTACTGTGCTACCCCCGCCAAGTCCGAGGGCGCGCCGgcacaccccggccgtcccagagcagtgcccacacagtgcgacgtcccccccaacagccgcttcgattgcgcccctgacaaggccatcacccaggaacagtgcgaggcccgcggctgctgctacatccctgcaaagcaggggctgcagggagcccagatggggcagccctggtgcttcttcccacccagctaccccagctacaagctggagaacctgagctcctctgaaatgggctacacggccaccctgacccgtaccacccccaccttcttccccaaggacatcctgaccctgcggctggacgtgatgatggagactgagaaccgcctccacttcacgatcaaagatccagctaacaggcgctacgaggtgcccttggagaccccgcgtgtccacagccgggcaccgtccccactctacagcgtggagttctctgaggagcccttcggggtgatcgtgcaccggcagctggacggccgcgtgctgctgaacacgacggtggcgcccctgttctttgcggaccagttccttcagctgtccacctcgctgccctcgcagtatatcacaggcctcgccgagcacctcagtcccctgatgctcagcaccagctggaccaggatcaccctgtggaaccgggaccttgcgcccacgcccggtgcgaacctctacgggtctcaccctttctacctggcgctggaggacggcgggtcggcacacggggtgttcctgctaaacagcaatgccatggatgtggtcctgcagccgagccctgcccttagctggaggtcgacaggtgggatcctggatgtctacatcttcctgggcccagagcccaagagcgtggtgcagcagtacctggacgttgtgggatacccgttcatgccgccatactggggcctgggcttccacctgtgccgctggggctactcctccaccgctatcacccgccaggtggtggagaacatgaccagggcccacttccccctggacgtccaatggaacgacctggactacatggactcccggagggacttcacgttcaacaaggatggcttccgggacttcccggccatggtgcaggagctgcaccagggcggccggcgctacatgatgatcgtggatcctgccatcagcagctcgggccctgccgggagctacaggccctagacgagggtctgcggaggggggttttcatcaccaacgagaccggccagccgtgattgggaaggtatggcccgggtccactgccttccccgacttcaccaaccccacagccctggcctggtgggaggacatggtggctgagttccatgaccaggtgcccttcgacggcatgtggattgacatgaacgagccttccaacttcatcaggggctctgaggacggctgccccaacaatgagctggagaacccaccctacgtgcctggggtggttggggggaccctccaggcggcaaccatctgtgcctccagccaccagtttctctccacacactacaacctgcacaacctctacggcctgaccgaagccatcgcctcccacagggcgctggtgaaggctcgggggacacgcccatttgtgatctcccgctcgacctttgctggccacggccgatacgccggccactggacgggggacgtgtggagctcctgggagcagctcgcctcctccgtgccagaaatcctgcagtttaacctgctgggggtgcctctggtcggggccgacgtctgcggcttcctgggcaacacctcagaggagctgtgtgtgcgctggacccagctggggccttctaccccttcatgcggaaccacaacagcctgctcagtctgccccaggagccgtacagcttcagcgagccggcccagcaggccatgaggaaggccctcaccctgcgctacgcactcctcccccacctctacacgctgttccaccaggcccacgtcgcgggggagaccgtggcccggcccctcttcctggagttccccaaggactctagcacctggactgtggaccaccagctcctgtggggggaggccctgctcatcaccccagtgctccaggccgggaaggccgaagtgactggctacttccccttgggcacatggtacgacctgcagacggtgccaatagaggcccttggcagcctcccacccccacctgcagctccccgtgagccagccatccacagcgaggggcagtgggtgacgctgccggcccccctggacaccatcaacgtccacctccgggctgggtacatcatccccctgcagggccctggcctcacaaccacagagtcccgccagcagcccatggccctggctgtggccctgaccaagggtggagaggcccgaggggagctgttctgggacgatggagagagcctggaagtgctggagcgaggggcctacacacaggtcatcttcctggccaggaataacacgatcgtgaatgagctggtacgtgtgaccagtgagggagctggcctgcagctgcagaaggtgatgtcctgggcgtggccacggcgccccagcaggtcctctccaacggtgtccctgtctccaacttcacctacagccccgacaccaaggtcctggacatctgtgtctcgctgttgatgggagagcagtttctcgtcagctggtgttagtctaga gcttgctagcggccgcConstruct 1460

The GILTΔ2-7/K40-GAA70-952 cassette below was cloned using the Asp718and NotI sites of the cassette and vector pCEP4 to producepCEP-GILTΔ2-7/K40-GAA70-952 (Plasmid p1460). Restriction sites forcloning are in lowercase bold. The spacer amino acid sequence Gly, Ala,Pro (underlined sequence) separate the GAA gene and GILTΔ2-7/K40 tag(upper case sequence). The spacer and tag are placed upstream of GAAresidue Ala70. The GILTΔ2-7/K40 cassette contains an Arg to Lyssubstitution at amino acid 40 of the human IGF-II sequence (uppercasebold).

(SEQ ID NO: 13) ggtaccagctgctagcaagctattcacaccaATGGGAATCCCAATGGGGAAGTCGATGCTGGTGCTTCTCACCTTCTTGGCCTTCGCCTCGTGCTGCATTGCTGCTCTGTGCGGCGGGGAGCTGGTGGACACCCTCCAGTTCGTCTGTGGGGACCGCGGCTTCTACTTCAGCAGGCCCGCAAGCCGTGTGAGCCGTCGCAGCAAGGGCATCGTTGAGGAGTGCTGTTTCCGCAGCTGTGACCTGGCCCTCCTGGAGACGTACTGTGCTACCCCCGCCAAGTCCGAGGGCGCGCCGgcacaccccggccgtcccagagcagtgcccacacagtgcgacgtcccccccaacagccgcttcgattgcgcccctgacaaggccatcacccaggaacagtgcgaggcccgcggctgctgctacatccctgcaaagcaggggctgcagggagcccagatggggcagccctggtgcttcttcccacccagctaccccagctacaagctggagaacctgagctcctctgaaatgggctacacggccaccctgacccgtaccacccccaccttcttccccaaggacatcctgaccctgcggctggacgtgatgatggagactgagaaccgcctccacttcacgatcaaagatccagctaacaggcgctacgaggtgcccttggagaccccgcgtgtccacagccgggcaccgtccccactctacagcgtggagttctctgaggagcccttcggggtgatcgtgcaccggcagctggacggccgcgtgctgctgaacacgacggtggcgcccctgttctttgcggaccagttccttcagctgtccacctcgctgccctcgcagtatatcacaggcctcgccgagcacctcagtcccctgatgctcagcaccagctggaccaggatcaccctgtggaaccgggaccttgcgcccacgcccggtgcgaacctctacgggtctcaccctttctacctggcgctggaggacggcgggtcggcacacggggtgttcctgctaaacagcaatgccatggatgtggtcctgcagccgagccctgcccttagctggaggtcgacaggtgggatcctggatgtctacatcttcctgggcccagagcccaagagcgtggtgcagcagtacctggacgttgtgggatacccgttcatgccgccatactggggcctgggcttccacctgtgccgctggggctactcctccaccgctatcacccgccaggtggtggagaacatgaccagggcccacttccccctggacgtccaatggaacgacctggatacatggatcccggagggacttcacgttcaacaaggatggcttccgggacttcccggccatggtgcaggagctgcaccagggcggccggcgctacatgatgatcgtggatcctgccatcagcagctcgggccctgccgggagctacaggccctacgacgagggtctgcggagggggttttcatcaccaacgagaccggccagccgctgattgggaaggtatggcccgggtccactgccttccccgacttcaccaaccccacagccctggcctggtgggaggacatggtggctgagttccatgaccaggtgcccttcgacggcatgtggattgacatgaacgagccttccaacttcatcaggggctctgaggacggctgccccaacaatgagctggagaacccaccctacgtgcctggggtggttggggggaccctccaggcggcaaccatctgtgcctccagccaccagtttctctccacacactacaacctgcacaacctctacggcctgaccgaagccatcgcctcccacagggcgctggtgaaggctcgggggacacgcccatttgtgatctcccgctcgacctttgctggccacggccgatacgccggccactggacgggggacgtgtggagctcctgggagcagctcgcctcctccgtgccagaaatcctgcagtttaacctgctgggggtgcctctggtcggggccgacgtctgcggcttcctgggcaacacctcagaggagctgtgtgtgcgctggacccagctggggccttctaccccttcatgcggaaccacaacagcctgctcagtctgccccaggagccgtacagcttcagcgagccggcccagcaggccatgaggaaggccctcaccctgcgctacgcactcctcccccacctctacacgctgttccaccaggcccacgtcgcgggggagaccgtggcccggcccctcttcctggagttccccaaggactctagcacctggactgtggaccaccagctcctgtggggggaggccctgctcatcaccccagtgctccaggccgggaaggccgaagtgactggctacttccccttgggcacatggtacgacctgcvagacggtgccaatagaggcccttggcagcctcccacccccacctgcagctccccgtgagccagccatccacagcgaggggcagtgggtgacgctgccggcccccctggacaccatcaacgtccacctccgggctgggtacatcatccccctgcagggccctggcctcacaaccacagagtcccgccagcagcccatggccctggctgtggccctgaccaagggtggagaggcccgaggggagctgttctgggacgatggagagagcctggaagtgctggagcgaggggcctacacacaggtcatcttcctggccaggaataacacgatcgtgaatgagctggtacgtgtgaccagtgagggagctggcctgcagctgcagaaggtgactgtcctgggcgtggccacggcgccccagcaggtcctctccaacggtgtccctgtctccaacttcacctacagccccgacaccaaggtcctggacatctgtgtctcgctgttgatgggagagcagtttctcgtcagctggtggttagtctag agcttgctagcggccgcConstruct 1461

The GILTΔ2-7/A37-GAA70-952 cassette below was cloned using the Asp718and NotI sites of the cassette and vector pCEP4 to producepCEP-GILTΔ2-7/A37-GAA70-952 (Plasmid p1461). Restriction sites forcloning are in lowercase bold. The spacer amino acid sequence Gly, Ala,Pro (underlined sequence) separate the GAA gene and GILTΔ2-7/A37 tag(upper case sequence). The spacer and tag are placed upstream of GAAresidue Ala70. The GILTΔ2-7/A37 cassette contains an Arg to Alasubstitution at amino acid 37 of the human IGF-II sequence (uppercasebold).

(SEQ ID NO: 14) ggtaccagctgctagcaagctaattcacaccaATGGGAATCCCAATGGGGAAGTCGATGCTGGTGCTTCTCACCTTCTTGGCCTTCGCCTCGTGCTGCATTGCTGCTCTGTGCGGCGGGGAGCTGGTGGACACCCTCCAGTTCGTCTGTGGGGACCGCGGCTTCTACTTCAGCAGGCCCGCAAGCCGTGTGAGCGCTCGCAGCCGTGGCATCGTTGAGGAGTGCTGTTTCCGCAGCTGTGACCTGGCCCTCCTGGAGACGTACTGTGCTACCCCCGCCAAGTCCGAGGGCGCGCCGgcacaccccggccgtcccagagcagtgcccacacagtgcgacgtcccccccaacagccgcttcgattgcgcccctgacaaggccatcacccaggaacagtgcgaggcccgcggctgctgctacatccctgcaaagcaggggctgcagggagcccagatggggcagccctggtgcttcttcccacccagctaccccagctacaagctggagaacctgagctcctctgaaatgggctacacggccaccctgacccgtaccacccccaccttcttccccaaggacatcctgaccctgcggctggacgtgatgatggagactgagaaccgcctccacttcacgatcaaagatccagctaacaggcgctacgaggtgcccttggagaccccgcgtgtccacagccgggcaccgtccccactctacagcgtggagttctctgaggagcccttcggggtgatcgtgcaccggcagctggacggccgcgtgctgctgaacacgacggtggcgcccctgttctttgcggaccagttccttcagctgtccacctcgctgccctcgcagtatatcacaggcctcgccgagcacctcagtcccctgatgctcagcaccagctggaccaggatcaccctgtggaaccgggaccttgcgcccacgcccggtgcgaacctctacgggtctcaccctttctacctggcgctggaggacggcgggtcggcacacggggtgttcctgctaaacagcaatgccatggatgtggtcctgcagccgagccctgcccttagctggaggtcgacaggtgggatcctggatgtctacatcttcctgggcccagagcccaagagcgtggtgcagcagtacctggacgttgtgggatacccgttcatgccgccatactggggcctgggcttccacctgtgccgctggggctactcctccaccgctatcacccgccaggtggtggagaacatgaccagggcccacttccccctggacgtccaatggaacgacctggactacatggactcccggagggacttcacgttcaacaaggatggcttccgggacttcccggccatggtgcaggagctgcaccagggcggccggcgctacatgatgatcgtggatcctgccatcagcagctcgggccctgccgggagctacaggccctacgacgagggtctgcggaggggggttttcatcaccaacgagaccggccagccgctgattgggaaggtatggcccgggtccactgccttccccgacttcaccaaccccacagccctggcctggtgggaggacatggtggctgagttccatgaccaggtgcccttcgacggcatgtggattgacatgaacgagccttccaacttcatcaggggctctgaggacggctgccccaacaatgagctggagaacccaccctacgtgcctggggtggttggggggaccctccaggcggcaaccatctgtgcctccagccaccagtttctctccacacactacaacctgcacaacctctacggcctgaccgaagccatcgcctcccacagggcgctggtgaaggctcgggggacacgcccatttgtgatctcccgctcgacctttgctggccacggccgatacgccggccactggacgggggacgtgtggagctcctgggagcagctcgcctcctccgtgccagaaatcctgcagtttaacctgctgggggtgcctctggtcggggccgacgtctgcggcttcctgggcaacacctcagaggagctgtgtgtgcgctggacccagctgggggccttctaccccttcatgcggaaccacaacagcctgctcagtctgccccaggagccgtacagcttcagcgagccggcccagcaggccatgaggaaggccctcaccctgcgctacgcactcctcccccacctctacacgctgttccaccaggcccacgtcgcgggggagaccgtggcccggcccctcttcctggagttccccaaggactctagcacctggactgtggaccaccagctcctgtggggggaggccctgctcatcaccccagtgctccaggccgggaaggccgaagtgactggctacttccccttgggcacatggtacgacctgcagacggtgccaatagaggcccttggcagcctcccacccccacctgcagctccccgtgagccagccatccacagcgaggggcagtgggtgacgctgccggcccccctggacaccatcaacgtccacctccgggctgggtacatcatccccctgcagggccctggcctcacaaccacagagtcccgccagcagcccatggccctggctgtggccctgaccaagggtggagaggcccgaggggagctgttctgggacgatggagagagcctggaagtgctggagcgaggggcctacacacaggtcatcttcctggccaggaataacacgatcgtgaatgagctggtacgtgtgaccagtgagggagctggcctgcagctgcagaaggtgactgtcctgggcgtggccacggcgccccagcaggtcctctccaacggtgtccctgtctccaacttcacctacagccccgacaccaaggtcctggacatctgtgtctcgctgttgatgggagagcagtttctcgtcagctggtgttagtc tagagcttgctagcggccgcConstruct 1463

The GILTΔ2-7/A40-GAA70-952 cassette below was cloned using the Asp718and NotI sites of the cassette and vector pCEP4 to producepCEP-GILTΔ2-7/A40-GAA70-952 (Plasmid p1463). Restriction sites forcloning are in lowercase bold. The spacer amino acid sequence Gly, Ala,Pro (underlined sequence) separate the GAA gene and GILTΔ2-7/A40 tag(upper case sequence). The spacer and tag are placed upstream of GAAresidue Ala70. The GILTΔ2-7/A40 cassette contains an Arg to Alasubstitution at amino acid 40 of the human IGF2 sequence (uppercasebold).

(SEQ ID NO: 15) ggtaccagctgctagcaagctaattcacaccaATGGGAATCCCAATGGGGAAGTCGATGCTGGTGCTTCTCACCTTCTTGGCCTTCGCCTCGTGCTGCATTGCTGCTCTGTGCGGCGGGGAGCTGGTGGACACCCTCCAGTTCGTCTGTGGGGACCGCGGCTTCTACTTCAGCAGGCCCGCAAGCCGTGTGAGCCGTCGCAGCGCTGGCATCGTTGAGGAGTGCTGTTTCCGCAGCTGTGACCTGGCCCTCCTGGAGACGTACTGTGCTACCCCCGCCAAGTCCGAGGGCGCGCCGgcacaccccggccgtcccagagcagtgcccacacagtgcgacgtcccccccaacagccgcttcgattgcgcccctgacaaggccatcacccaggaacagtgcgaggcccgcggctgctgctacatccctgcaaagcaggggctgcagggagcccagatggggcagccctggtgcttcttcccacccagctaccccagctacaagctggagaacctgagctcctctgaaatgggctacacggccaccctgacccgtaccacccccaccttcttccccaaggacatcctgaccctgcggctggacgtgatgatggagactgagaaccgcctccacttcacgatcaaagatccagctaacaggcgctacgaggtgcccttggagaccccgcgtgtccacagccgggcaccgtccccactctacagcgtggagttctctgaggagcccttcggggtgatcgtgcaccggcagctggacggccgcgtgctgctgaacacgacggtggcgcccctgttctttgcggaccagttccttcagctgtccacctcgctgccctcgcagtatatcacaggcctcgccgagcacctcagtcccctgatgctcagcaccagctggaccaggatcaccctgtggaaccgggaccttgcgcccacgcccggtgcgaacctctacgggtctcaccctttctacctggcgctggaggacggcgggtcggcacacggggtgttcctgctaaacagcaatgccatggatgtggtcctgcagccgagccctgcccttagctggaggtcgacaggtgggatcctggatgtctacatcttcctgggcccagagcccaagagcgtggtgcagcagtacctggacgttgtgggatacccgttcatgccgccatactggggcctgggcttccacctgtgccgctggggctactcctccaccgctatcacccgccaggtggtggagaacatgaccagggcccacttccccctggacgtccaatggaacgacctggactacatggactcccggagggacttcacgttcaacaaggatggcttccgggacttcccggccatggtgcaggagctgcaccagggcggccggcgctacatgatgatcgtggatcctgccatcagcagctcgggccctgccgggagctacaggccctacgacgagggtctgcggaggggggttttcataccaacgagaccggccagccgctgattgggaaggtatggcccgggtccactgccttccccgacttcaccaaccccacagccctggcctggtgggaggacatggtggctgagttccatgaccaggtgcccttcgacggcatgtggattgacatgaacgagccttccaacttcatcaggggctctgaggacggctgccccaacaatgagctggagaacccaccctacgtgcctggggtggttggggggaccctccaggcggcaaccatctgtgcctccagccaccagtttctctccacacactacaacctgcacaacctctacggcctgaccgaagccatcgcctcccacagggcgctggtgaaggctcgggggacacgcccatttgtgatctcccgctcgacctttgctggccacggccgatacgccggccactggacgggggacgtgtggagctcctgggagcagctcgcctcctccgtgccagaaatcctgcagtttaacctgctgggggtgcctctggtcggggccgacgtctgcggcttcctgggcaacacctcagaggagctgtgtgtgcgctggacccagctgggggccttctaccccttcatgcggaaccacaacagcctgctcagtctgccccaggagccgtacagcttcagcgagccggcccagcaggccatgaggaaggccctcaccctgcgctacgcactcctcccccacctctacacgctgttccaccaggcccacgtcgcgggggagaccgtggcccggcccctcttcctggagttccccaaggactctagcacctggactgtggaccaccagctcctgtggggggaggccctgctcatcaccccagtgctccaggccgggaaggccgaagtgactggctacttccccttgggcacatggtacgacctgcagacggtgccaatagaggcccttggcagcctcccacccccacctgcagctccccgtgagccagccatccacagcgaggggcagtgggtgacgctgccggcccccctggacaccatcaacgtccacctccgggctgggtacatcatccccctgcagggccctggcctcacaaccacagagtcccgccagcagcccatggccctggctgtggccctgaccaagggtggagaggcccgaggggagctgttctgggacgatggagagagcctggaagtgctggagcgaggggcctacacacaggtcatcttcctggccaggaataacacgatcgtgaatgagctggtacgtgtgaccagtgagggagctggcctgcagctgcagaaggtgactgtcctgggcgtggccacggcgccccagcaggtcctctccaacggtgtccctgtctccaacttcacctacagccccgacaccaaggtcctggacatctgtgtctcgctgttgatgggagagcagtttctcgtcagctggtgttagtct agagcttgctagcggccgcConstruct 1479

The GILTΔ2-7M1/K37-GAA70-952 cassette below was cloned using the Asp718and NotI sites of the cassette and vector pCEP4 to producepCEP-GILTΔ2-7M1/K37-GAA70-952 (Plasmid p1479). Restriction sites forcloning are in lowercase bold. The spacer amino acid sequence Gly, Ala,Pro (underlined sequence) separate the GAA gene and GILTΔ2-7M1/K37 tag(upper case sequence). The spacer and tag are placed upstream of GAAresidue Ala70. The GILTΔ2-7M1/K37 cassette contains an Arg to Lyssubstitution at amino acid 37 of the human IGF-II sequence (uppercasebold).

(SEQ ID NO: 16) ggtaccaagcttgccATGGGAATCCCAATGGGCAAGTCGATGCTGGTGCTGCTCACCTTCTTGGCCTTTGCCTCGTGCTGCATTGCCGCTCTGTGCGGCGGGGAACTGGTGGACACCCTCCAATTCGTCTGTGGGGACCGGGGCTTCTACTTCAGCAGACCCGCAAGCCGTGTGAGTAAGCGCAGCCGTGGCATTGTTGAGGAGTGCTGTTTTCGCAGCTGTGACCTGGCTCTCCTGGAGACGTACTGCGCTACCCCCGCCAAGTCTGAGGGCGCGCCGgcacaccccggccgtcccagagcagtgcccacacagtgcgacgtcccccccaacagccgcttcgattgcgcccctgacaggccatcacccaggaacagtgcgaggccgcggctgctgctacatccctgcaaagcaggggctgcagggagcccagatggggcagccctggtgcttcttcccacccagctaccccagctacaagctggagaacctgagctcctctgaaatgggctacacggccaccctgacccgtaccacccccaccttcttccccaaggacatcctgaccctgcggctggacgtgatgatggagactgagaaccgcctccacttcacgatcaaagatccagctaacaggcgctacgaggtgcccttggagaccccgcgtgtccacagccgggcaccgtccccactctacagcgtggagttctctgaggagcccttcggggtgatcgtgcaccggcagctggacggccgcgtgctgctgaacacgacggtggcgcccctgttctttgcggaccagttccttcagctgtccacctcgctgccctcgcagtatatcacaggcctcgccgagcacctcagtcccctgatgctcagcaccagctggaccaggatcaccctgtggaaccgggaccttgcgcccacgcccggtgcgaacctctacgggtctcaccctttctacctggcgctggaggacggcgggtcggcacacggggtgttcctgctaaacagcaatgccatggatgtggtcctgcagccgagccctgccatagaggaggtcgacaggtgggatcctggatgtctacatcttcctgggcccagagcccaagagcgtggtgcagcagtacctggacgttgtgggatacccgttcatgccgccatactggggcctgggcttccacctgtgccgctggggctactcctccaccgctatcacccgccaggtggtggagaacatgaccagggcccacttccccctggacgtccaatggaacgacctggactacatggactcccggagggacttcacgttcaacaaggatggcttccgggacttcccggccatggtgcaggagctgcaccagggcggccggcgctacatgatgatcgtggatcctgccatcagcagctcgggccagccgggagctacaggccctacgacgagggtctgcggaggggggttttcatcaccaacgagaccggccagccgctgattgggaaggtatggcccgggtccactgccttccccgacttcaccaaccccacagccaggcctggtgggaggacatggtggctgagttccatgaccaggtgcccttcgacggcatgtggattgacatgaacgagccttccaacttcatcaggggctctgaggacggctgccccaacaatgagaggagaacccaccctacgtgcctggggtggttggggggaccctccaggcggcaaccatctgtgcctccagccaccagtttctctccacacactacaacctgcacaacctctacggcctgaccgaagccatcgcctcccacagggcgaggtgaaggctcgggggacacgcccatttgtgatctcccgctcgacctttgaggccacggccgatacgccggccactggacgggggacgtgtggagctcctgggagcagctcgcctcctccgtgccagaaatcctgcagtttaacctgctgggggtgcctctggtcggggccgacgtctgcggcttcctgggcaacacctcagaggagagtgtgtgcgctggacccagctgggggccttctaccccttcatgcggaaccacaacagcctgctcagtagccccaggagccgtacagcttcagcgagccggcccagcaggccatgaggaaggccctcaccctgcgctacgcactcctcccccacctctacacgctgttccaccaggcccacgtcgcgggggagaccgtggcccggcccctcttcctggagttccccaaggactctagcacctggactgtggaccaccagctcctgtggggggaggccctgctcatcaccccagtgctccaggccgggaaggccgaagtgactggctacttccccttgggcacatggtacgacctgcagacggtgccaatagaggcccttggcagcctcccacccccacctgcagctccccgtgagccagccatccacagcgaggggcagtgggtgacgctgccggcccccctggacaccatcaacgtccacctccgggctgggtacatcatccccctgcagggccaggcctcacaaccacagagtcccgccagcagcccatggccctggctgtggccctgaccaagggtggagaggcccgaggggagagttctgggacgatggagagagcctggaagtgctggagcgaggggcctacacacaggtcatcttcctggccaggaataacacgatcgtgaatgagctggtacgtgtgaccagtgagggagctggcctgcagctgcagaaggtgactgtcctgggcgtggccacggcgccccagcaggtcctctccaacggtgtccctgtctccaacttcacctacagccccgacaccaaggtcctggacatctgtgtctcgctgttgatgggagagcagtttctcgtcagctggtgttagtctagagcttgctagcggccgcConstruct 1487

The GILTΔ2-7M1/A37-GAA70-952 cassette below was cloned using the Asp718and NotI sites of the cassette and vector pCEP4 to producepCEP-GILTΔ2-7M1/A37-GAA70-952 (Plasmid p1487). Restriction sites forcloning are in lowercase bold. The spacer amino acid sequence Gly, Ala,Pro (underlined sequence) separate the GAA gene and GILTΔ2-7M1/A37 tag(upper case sequence). The spacer and tag are placed upstream of GAAresidue Ala70. The GILTΔ2-7M1/A37 cassette contains an Arg to Alasubstitution at amino acid 37 of the human IGF-II sequence (uppercasebold).

(SEQ ID NO: 17) ggtaccaagcttgccATGGGAATCCCAATGGGCAAGTCGATGCTGGTGCTGCTCACCTTCTTGGCCTTTGCCTCGTGCTGCATTGCCGCTCTGTGCGGCGGGGAACTGGTGGACACCCTCCAATTCGTCTGTGGGGACCGGGGCTTCTACTTCAGCAGACCCGCAAGCCGTGTGAGTGCTCGCAGCCGTGGCATTGTTGAGGAGTGCTGTTTTCGCAGCTGTGACCTGGCTCTCCTGGAGACGTACTGCGCTACCCCCGCCAAGTCTGAGGGCGCGCCGgcacaccccggccgtcccagagcagtgcccacacagtgcgacgtcccccccaacagccgcttcgattgcgcccctgacaaggccatcacccaggaacagtgcgaggcccgcggctgctgctacatccctgcaaagcaggggctgcagggagcccagatggggcagccctggtgcttcttcccacccagctaccccagctacaagctggagaacctgagctcctctgaaatgggctacacggccaccctgacccgtaccacccccaccttcttccccaaggacatcctgaccctgcggctggacgtgatgatggagactgagaaccgcctccacttcacgatcaaagatccagctaacaggcgctacgaggtgcccttggagaccccgcgtgtccacagccgggcaccgtccccactctacagcgtggagttctagaggagccatcggggtgatcgtgcaccggcagctggacggccgcgtgctgctgaacacgacggtggcgcccctgactttgcggaccagaccttcagctgtccacctcgctgccacgcagtatatcacaggcctcgccgagcacctcagtcccctgatgctcagcaccagctggaccaggatcaccagtggaaccgggaccttgcgcccacgcccggtgcgaacctctacgggtctcaccctttctacctggcgctggaggacggcgggtcggcacacggggtgacctgctaaacagcaatgccatggatgtggtcctgcagccgagccctgcccttagaggaggtcgacaggtgggatcctggatgtctacatcttcctgggcccagagcccaagagcgtggtgcagcagtacctggacgttgtgggatacccgttcatgccgccatactggggcctgggcttccacctgtgccgctggggctactcaccaccgctatcacccgccaggtggtggagaacatgaccagggcccacttccccctggacgtccaatggaacgacctggactacatggactcccggagggacttcacgttcaacaaggatggcttccgggacttcccggccatggtgcaggagctgcaccagggcggccggcgctacatgatgatcgtggatcctgccatcagcagacgggccctgccgggagctacaggccctacgacgagggtctgcggaggggggttttcatcaccaacgagaccggccagccgctgattgggaaggtatggcccgggtccactgccttccccgacttcaccaaccccacagccctggcctggtgggaggacatggtggctgagaccatgaccaggtgccatcgacggcatgtggattgacatgaacgagccttccaacttcatcaggggctctgaggacggctgccccaacaatgagctggagaacccaccctacgtgcctggggtggttggggggaccctccaggcggcaaccatctgtgcctccagccaccagtttactccacacactacaacctgcacaacctctacggcctgaccgaagccatcgcctcccacagggcgctggtgaaggctcgggggacacgcccatttgtgatctcccgctcgacctttgctggccacggccgatacgccggccactggacgggggacgtgtggagctcctgggagcagctcgcctcctccgtgccagaaatcctgcagtttaacctgctgggggtgcctctggtcggggccgacgtctgcggcttcctgggcaacacctcagaggagagtgtgtgcgaggacccagagggggccttctaccccttcatgcggaaccacaacagcctgctcagtctgccccaggagccgtacagcttcagcgagccggcccagcaggccatgaggaaggccctcaccagcgctacgcactcctcccccacctctacacgctgttccaccaggcccacgtcgcgggggagaccgtggcccggcccctcttcctggagttccccaaggactctagcacctggactgtggaccaccagacctgtggggggaggccagctcatcaccccagtgctccaggccgggaaggccgaagtgactggctacttcccatgggcacatggtacgacctgcagacggtgccaatagaggccatggcagcctcccacccccacctgcagctccccgtgagccagccatccacagcgaggggcagtgggtgacgctgccggcccccctggacaccatcaacgtccacctccgggctgggtacatcatccccctgcagggccaggcctcacaaccacagagtcccgccagcagcccatggccaggctgtggccctgaccaagggtggagaggcccgaggggagctgttctgggacgatggagagagcctggaagtgctggagcgaggggcctacacacaggtcatatcctggccaggaataacacgatcgtgaatgagaggtacgtgtgaccagtgagggagctggcctgcagctgcagaaggtgactgtcctgggcgtggccacggcgccccagcaggtcctctccaacggtgtccagtaccaacttcacctacagccccgacaccaaggtcctggacatctgtgtacgctgttgatgggagagcagtttctcgtcagctggtgttagtctagagcttgctagcggccgc

As shown in FIG. 3, three exemplary mutants (i.e., constructs 1459, 1460and 1461) in which alanine or lysine has been substituted for one of thecanonical arginine residues were expressed without detectable cleavageby furin. As also shown in FIG. 3 (right panel), construct 1461containing a R37A substitution is additionally resistant to addition ofexogenous furin.

Construct 1726

The GILTΔ2-7Δ30-39-GAA70-952 cassette below was cloned using the Asp718and NotI sites of the cassette and vector pCEP4 to producepCEP-GILTΔ2-7Δ30-39-GAA70-952 (Plasmid 1726). Restriction sites forcloning are in lowercase bold. The spacer amino acid sequence Gly, Ala,Pro (underlined sequence) separate the GAA gene and GILTΔ2-7Δ30-39 tag(upper case sequence). The spacer and tag are placed upstream of GAAresidue Ala70. The GILTΔ2-7Δ30-39 cassette contains a deletion of aminoacid residues 30-39 (Arg-Pro-Ala-Ser-Arg-Val-Ser-Arg-Arg-Ser) from thehuman IGF-II sequence.

(SEQ ID NO: 18) ggtaccagctgctagcaagctaattcacaccaATGGGAATCCCAATGGGGAAGTCGATGCTGGTGCTTCTCACCTTCTTGGCCTTCGCCTCGTGCTGCATTGCTGCTCTGTGCGGCGGGGAGCTGGTGGACACCCTCCAGTTCGTCTGTGGGGACCGCGGCTTCTACTTCAGCCGTGGCATCGTCCCCGCCAAGTCCGAGGGCGCGCCGgcacaccccggccgtcccagagcagtgcccacacagtgcgacgtcccccccaacagccgcttcgattgcgccctgacaaggccatcacccaggaacagtgcgaggcccgcggctgctgctacatccctgcaaagcaggggctgcagggagcccagatggggcagccctggtgcttcttcccacccagctaccccagctacaagctggagaacctgagctcctctgaaatgggcatacacggccaccctgacccgtaccacccccaccttcttccccaggacatcctgaccctgcggctggacgtgatgatggagactgagaaccgcctccacttcacgatcaaagatccagctaacaggcgctacgaggtgcccttggagaccccgcgtgtccacagccgggcaccgtcccactctacagcgtggagttctctgaggagcccttcggggtgatcgtgcaccggcagctggacggccgcgtgctgctgaacacgacggtggcgcccctgttctttgcggaccagttccttcagctgtccacctcgtgccctcgcagtatatcacaggcctcgccgagcacctcagtcccctgatcgtcagcaccagctggaccaggatcaccctgtggaaccgggacttgcgcccacgcccggtgcgaacctctacgggtctcaccctttctacctggaggtcagacaggtgggatcctggatgtctacatcttcctgggcccagagcccaagagcgtggtgcagcagtacctggacgccttagctggaggtcgacaggtgggatcctggatgtctacatcttcctgggcccagagcccaagagcgtggtgcagcagtacctggacgttgtgggatacccgttcatgccgccatactggggcctgggcttccacctgtgccgctggggctactcctccaccgctatcacccgccaggtggtggagaacatgaccagggcccacttccccctggacgtccaatggaacgacctggactacatggactcccggagggacttcacgttcaacaaggatggcttccgggacttcccggccatggtgcaggagctgcaccagggcggccggcgctacatgatgatcgtggatcctgccatcagcagctcgggccctgccgggagctacaggccctacgacgagggtctgcggaggggggttttcatcaccaacgagaccggccagccgctgattgggaggtatggcccgggtccactgccttccccgacttcaccaaccccacagccctggcctggtgggaggacatggtggctgagttccatgaccaggtgcccttcgacggcatgtggattgacatgaacgagccttccaacttcatcaggggctctgaggacggctgccccaacaatgagctggagaacccaccctacgtgcctggggtggtggggggaccctccaggcggcaaccatctgtgcctccagccaccagtttctctccacacactacaacctgcacaacctctacggcctgaccgaagccatcgcctcccacagggcgctggtgaaggctccgggggacacgcccatttgtgatctcccgctcgacctttgctggccacggccgatacgccggccactggacgggggacgtgtggagctcctgggagcagctcgcctcctccgtgccagaaatcctgcagtttaacctgctgggggtgcctctggtcggggccgacgtctgcggcttcctgggcaacacctcagaggagctgtgtgcgctggacccagctgggggccttctaccccttcatgcggaaccacaacagcctgctcagtgtgccccaggagccgtacagcttcagcgagccggcccagcaggccatgaggaaggccctcaccctgcgctacgcactcctcccccacctctacacgctgttccaccaggcccacgtcgcgggggagaccgtggcccggcccctcttcctggagttccccaggactctagcacctggactgtggaccaccagctcctgtggggggaggccctgctcatcaccccagtgctccaggccgggaaggccgaagtgactggctacttcccctgggcacatggtacgacctgcagacggtgccaatagaggcccttggcagcctcccacccccacctgcagctccccgtgagccagccatccacgcgaggggcagtgggtgacgctgccgcccccctggacaccatcaacgtccacctccgggctgggtacatcatccccctgcagggccctggcctcacaaccacagagtcccgccagcagcccatggccctggctgtggccctgaccaagggtggagaggcccgaggggagctgttctgggacgatggagagagcctggaagtgctggagcgaggggcctacacacaggtcatcttcctggccaggaataacacgatcgtgaatgagctggtacgtgtgaccagtgagggagctggccatgcagctgcagaaggtgactgtcctgggcgtggccacggcgccccagcaggtcctctccaacggtgtccctgtctccaacttcacctacagccccgacaccaaggtcctggacatctgtgtctcgctgttgatgggagagcagtttctcgtcagctggtgttagt ctagagcttgctagcggccgcConstruct 1749

The GILTΔ2-7Δ31-39-GAA70-952 cassette below was cloned using the Asp718and NotI sites of the cassette and vector pCEP4 to producepCEP-GILTΔ2-7Δ31-39-GAA70-952 (Plasmid 1749). Restriction sites forcloning are in lowercase bold. The spacer amino acid sequence Gly, Ala,Pro (underlined sequence) separate the GAA gene and GILTΔ2-7Δ31-39 tag(upper case sequence). The spacer and tag are placed upstream of GAAresidue Ala70. The GILTΔ2-7Δ31-39 cassette contains a deletion of aminoacid residues 31-39 (Pro-Ala-Ser-Arg-Val-Ser-Arg-Arg-Ser) from the humanIGF-II sequence.

(SEQ ID NO: 19) ggtaccagcgctagcaagctaattcacaccaATGGGAATCCCAATGGGGAAGTCGATGCTGGTGCTTCTCACCTTCTTGGCCTTCGCCTCGTGCTGCATTGCTGCTCTGTGCGGCGGGGAGCTGGTGGACACCCTCCAGTTCGTCTGTGGGGACCGCGGCTTCTACTTCAGCAGGCGTGGCATCGTTGAGGAGTGCTGTTTCCGCAGCTGTGACCTGGCCCTCCTGGAGACGTACTGTGCTACCCCCGCCAAGTCCGAGGGCGCGCCGgcacaccccggccgtcccagagcagtgcccacacagtgcgacgtcccccccaacagccgcttcgattgcgcccctgacaaggccatcacccaggaacagtgcgaggcccgcggctgctgctacatccctgcaaagcaggggctgcagggagcccagatggggcagccctggtgcttatcccacccagctaccccagctacaagaggagaacctgagctcctctgaaatgggctacacggccaccctgacccgtaccacccccaccttatccccaaggacatcctgaccctgcggctggacgtgatgatggagactgagaaccgcctccacttcacgatcaaagatccagctaacaggcgctacgaggtgcccttggagaccccgcgtgtccacagccgggcaccgtccccactctacagcgtggagttctctgaggagccatcggggtgatcgtgcaccggcagctggacggccgcgtgctgctgaacacgacggtggcgcccctgttctttgcggaccagttccttcagctgtccacctcgctgccctcgcagtatatcacaggcctcgccgagcacctcagtcccctgatgacagcaccagctggaccaggatcaccagtggaaccgggaccttgcgcccacgcccggtgcgaacctctacgggtctcaccctttctacctggcgctggaggacggcgggtcggcacacggggtgttcctgctaaacagcaatgccatggatgtggtcctgcagccgagccctgcccttagctggaggtcgacaggtgggatcctggatgtctacatcttcctgggcccagagcccaagagcgtggtgcagcagtacctggacgagtgggatacccgttcatgccgccatactggggcctgggcttccacctgtgccgctggggctactcctccaccgctatcacccgccaggtggtggagaacatgaccagggcccacttccccctggacgtccaatggaacgacctggactacatggactcccggagggacttcacgttcaacaaggatggatccgggacttcccggccatggtgcaggagagcaccagggcggccggcgctacatgatgatcgtggatcctgccatcagcagctcgggccctgccgggagctacaggccctacgacgagggtctgcggaggggggttttcatcaccaacgagaccggccagccgctgattgggaaggtatggcccgggtccactgccttccccgacttcaccaaccccacagccctggcctggtgggaggacatggtggctgagttccatgaccaggtgcccttcgacggcatgtggattgacatgaacgagccttccaacttcatcaggggctctgaggacggctgccccaacaatgagaggagaacccaccctacgtgcctggggtggttggggggaccaccaggcggcaaccatctgtgcctccagccaccagtttactccacacactacaacctgcacaacctctacggcctgaccgaagccatcgcctcccacagggcgctggtgaaggctcgggggacacgcccatttgtgatctcccgctcgacctttgctggccacggccgatacgccggccactggacgggggacgtgtggagctcctgggagcagctcgcctcctccgtgccagaaatcctgcagtttaacctgctgggggtgcctctggtcggggccgacgtctgcggcttcctgggcaacacctcagaggagctgtgtgtgcgctggacccagctgggggccttctaccccttcatgcggaaccacaacagcctgctcagtctgccccaggagccgtacagcttcagcgagccggcccagcaggccatgaggaaggccacaccctgcgctacgcactcctcccccacctctacacgctgttccaccaggcccacgtcgcgggggagaccgtggcccggcccctcttcctggagttccccaaggactctagcacctggactgtggaccaccagctcctgtggggggaggccctgctcatcaccccagtgctccaggccgggaaggccgaagtgactggctacttccccttgggcacatggtacgacctgcagacggtgccaatagaggcccttggcagcctcccacccccacctgcagctccccgtgagccagccatccacagcgaggggcagtgggtgacgctgccggcccccctggacaccatcaacgtccacctccgggctgggtacatcatccccctgcagggccaggcctcacaaccacagagtmcgccagcagcccatggccaggctgtggccctgaccaagggtggagaggcccgaggggagctgttctgggacgatggagagagcctggaagtgctggagcgaggggcctacacacaggtcatcttcctggccaggaataacacgatcgtgaatgagaggtacgtgtgaccagtgagggagaggcctgcagctgcagaaggtgactgtcctgggcgtggccacggcgccccagcaggtcctctccaacggtgtccctgtctccaacttcacctacagccccgacaccaaggtcctggacatctgtgtctcgctgttgatgggagagcagtttctcgtcagaggtgttagtctagagcttgctagcggccgcConstruct 1746

The GILTΔ2-7Δ32-39-GAA70-952 cassette below was cloned using the Asp718and NotI sites of the cassette and vector pCEP4 to producepCEP-GILTΔ2-7Δ32-39-GAA70-952 (Plasmid 1746). Restriction sites forcloning are in lowercase bold. The spacer amino acid sequence Gly, Ala,Pro (underlined sequence) separate the GAA gene and GILTΔ2-7Δ32-39 tag(upper case sequence). The spacer and tag are placed upstream of GAAresidue Ala70. The GILTΔ2-7Δ32-39 cassette contains a deletion of aminoacid residues 32-39 (Ala-Ser-Arg-Val-Ser-Arg-Arg-Ser) from the humanIGF-II sequence.

(SEQ ID NO: 20) ggtaccagctgctagcaagctaattcacaccaATGGGAATCCCAATGGGGAAGTCGATGCTGGTGCTTCTCACCTTCTTGGCCTTCGCCTCGTGCTGCATTGCTGCTCTGTGCGGCGGGGAGCTGGTGGACACCCTCCAGTTCGTCTGTGGGGACCGCGGCTTCTACTTCAGCAGGCCCCGTGGCATCGTTGAGGAGTGCTGTTTCCGCAGCTGTGACCTGGCCCTCCTGGAGACGTACTGTGCTACCCCCGCCAAGTCCGAGGGCGCGCCGgcacaccccggccgtcccagagcagtgcccacacagtgcgacgtcccccccaacagccgcttcgattgcgcccctgacaaggccatcacccaggaacagtgcgaggcccgcggctgctgctacatccctgcaaagcaggggctgcagggagcccagatggggcagccctggtgcttcttcccacccagctaccccagctacaagctggagaacctgagctcctctgaaatgggctacacggccaccctgacccgtaccacccccaccttcttccccaaggacatcctgaccctgcggctggacgtgatgatggagactgagaaccgcctccacttcacgatcaaagatccagctaacaggcgctacgaggtgcccttggagaccccgcgtgtccacagccgggcaccgtccccactctacagcgtggagttctctgaggagcccttcggggtgatcgtgcaccggcagctggacggccgcgtgctgctgaacacgacggtggcgcccctgttctttgcggaccagttccttcagctgtccacctcgctgccctcgcagtatatcacaggcctcgccgagcacctcagtcccctgatgctcagcaccagctggaccaggatcaccctgtggaaccgggaccttgcgcccacgcccggtgcgaacctctacgggtctcaccctactacctggcgctggaggacggcgggtcggcacacggggtgttcctgctaaacagcaatgccatggatgtggtcctgcagccgagccctgccatagctggaggtcgacaggtgggatcctggatgtctacatcttcctgggcccagagcccaagagcgtggtgcagcagtacctggacgttgtgggatacccgttcatgccgccatactggggcctgggcaccacctgtgccgctggggctactcctccaccgctatcacccgccaggtggtggagaacatgaccagggcccacttccccctggacgtccaatggaacgacctggactacatggactcccggagggacttcacgttcaacaaggatggcttccgggacttcccggccatggtgcaggagctgcaccagggcggccggcgctacatgatgatcgtggatcctgccatcagcagctcgggccctgccgggagctacaggccctacgacgagggtctgcggaggggggttttcatcaccaacgagaccggccagccgctgattgggaaggtatggcccgggtccactgccttccccgacttcaccaaccccacagccctggcctggtgggaggacatggtggctgagttccatgaccaggtgccatcgacggcatgtggattgacatgaacgagccttccaacttcatcaggggctctgaggacggctgccccaacaatgagaggagaacccaccctacgtgcctggggtggaggggggaccctccaggcggcaaccatctgtgcctccagccaccagtttctctccacacactacaacctgcacaacctctacggcctgaccgaagccatcgcctcccacagggcgctggtgaaggctcgggggacacgcccatttgtgatctcccgctcgacctttgaggccacggccgatacgccggccactggacgggggacgtgtggagctcctgggagcagctcgcctcctccgtgccagaaatcctgcagtttaacctgctgggggtgcctaggtcggggccgacgtctgcggcttcctgggcaacacctcagaggagagtgtgtgcgctggacccagctgggggccttctaccccttcatgcggaaccacaacagcctgacagtctgccccaggagccgtacagatcagcgagccggcccagcaggccatgaggaaggccctcaccctgcgctacgcactcctcccccacctctacacgctgttccaccaggcccacgtcgcgggggagaccgtggcccggcccctcttcctggagttccccaaggactctagcacctggactgtggaccaccagctcctgtggggggaggccctgacatcaccccagtgaccaggccgggaaggccgaagtgactggctacttccccttgggcacatggtacgacctgcagacggtgccaatagaggcccttggcagcctcccacccccacctgcagctccccgtgagccagccatccacagcgaggggcagtgggtgacgctgccggcccccctggacaccatcaacgtccacctccgggagggtacatcatccccctgcagggccctggcctcacaaccacagagtcccgccagcagcccatggccctggagtggccctgaccaagggtggagaggcccgaggggagagttctgggacgatggagagagcctggaagtgctggagcgaggggcctacacacaggtcatatcctggccaggaataacacgatcgtgaatgagaggtacgtgtgaccagtgagggagaggcctgcagagcagaaggtgactgtcctgggcgtggccacggcgccccagcaggtcctctccaacggtgtccctgtctccaacttcacctacagccccgacaccaaggtcctggacatctgtgtctcgctgttgatgggagagcagtttctcgtcagctggtgttagtctagagcttgctagcggccgcConstruct 1747

The GILTΔ2-7Δ33-39-GAA70-952 cassette below was cloned using the Asp718and NotI sites of the cassette and vector pCEP4 to producepCEP-GILTΔ2-7Δ33-39-GAA70-952 (Plasmid 1747). Restriction sites forcloning are in lowercase bold. The spacer amino acid sequence Gly, Ala,Pro (underlined sequence) separate the GAA gene and GILTΔ2-7Δ33-39 tag(upper case sequence). The spacer and tag are placed upstream of GAAresidue Ala70. The GILTΔ2-7Δ33-39 cassette contains a deletion of aminoacid residues 33-39 (Ser-Arg-Val-Ser-Arg-Arg-Ser) from the human IGF-IIsequence.

(SEQ ID NO: 21) ggtaccagctgctagcaagctaattcacaccaATGGGAATCCCAATGGGGAAGTCGATGCTGGTGCTTCTCACCTTCTTGGCCTTCGCCTCGTGCTGCATTGCTGCTCTGTGCGGCGGGGAGCTGGTGGACACCCTCCAGTTCGTCTGTGGGGACCGCGGCTTCTACTTCAGCAGGCCCGCACGTGGCATCGTTGAGGAGTGCTGTTTCCGCAGCTGTGACCTGGCCCTCCTGGAGACGTACTGTGCTACCCCCGCCAAGTCCGAGGGCGCGCCGgcacaccccggccgtcccagagcagtgcccacacagtgcgacgtcccccccaacagccgcttcgattgcgcccctgacaaggccatcacccaggaacagtgcgaggcccgcggctgctgctacatccctgcaagcaggggctgcagggagcccagatggggcagccctggtgcttcttcccacccagctaccccagctacaagctggagaacctgagctcctctgaaatgggctacacggccaccctgacccgtaccacccccaccttcttccccaaggacatcctgaccctgcggctggacgtgatgatggagactgagaaccgcctccacttcacgatcaaagatccagctaacaggcgctacgaggtgcccttggagaccccgcgtgtccacagccgggcaccgtccccactctacagcgtggagttctctgaggagcccttcggggtgatcgtgcaccggcagctggacggccgcgtgctgctgaacacgacggtggcgcccctgttctttgcggaccagttccttcagctgtccacctcgctgccctcgcagtatatcacaggcctcgccgagcacctcagtcccctgatgctcagcaccagctggaccaggatcaccctgtggaaccgggaccttgcgcccacgcccggtgcgaacctctacgggtctcaccctttctacctggcgctggaggacggcgggtcggcacacggggtgttcctgctaaacagcaatgccatggaatgtggtcctgcagccgagccctgcccttagctggaggtcgacaggtgggatcctggatgtctacatcttcctgggcccagagcccaagagcgtggtgcagcagtacctggacgttgtgggatacccgttcatgccgccatactggggcctgggcttccacctgtgccgctggggctactcctccaccgctatcacccgccaggtggtggagaacatgaccagggcccacttccccctggacgtccaatggaacgacctggactacatggactcccggagggacttcacgttcaacaaggatggcttccgggacttcccggccatggtgcaggagctgcaccagggcggccggcgctacatgatgatcgtggatcctgccatcagcagctcgggccctgccgggagctacaggccctacgacgagggtctgcggaggggggttttcatcaccaacgagaccggccagccgctgattgggaaggtatggcccgggtccactgccttccccgacttcaccaaccccacagccctggcctggtgggaggacatggtggctgagttccatgaccaggtgcccttcgacggcatgtggattgacatgaacgagccttccaacttcatcaggggctctgaggacggctgccccaacaatgagctggagaacccaccctacgtgcctggggtggtggggggaccctccaggcggcaaccatctgtgcctccagccaccagtttctctccacacactacaacctgcacaacctctacggcctgaccgaagccatcgcctcccacaggggcgctggtgaaggctcgggggacacgcccatttctgatctcccgctcgacctttgctggccacggccgatacgccggccactggacgggggacgtgtggagctcctgggagcagctcgcctcctccgtgccagaaatcctgcagtttaacctgctgggggtgcctctggtcggggccgacgtctgcggcttcctgggcaacacctcagaggactgtgtgtgcgctggacccagctgggggccttctaccccttcatgcggaccacaacagcctgctcagctggccccaggagccgtacagcttcagcgagccggcccagcaggccatgaggaaggccctcaccctgcgctacgcactcctcccccacctctacacgctgttccaccaggcccacgtgcgggggagaccgtggcccggcccctcttcctggagttccccaaggactctagcacctggactgtggaccaccagctcctgtggggggaggccctgctcatcaccccagtgctccaggccgggaaggccgaagtgactggctacttccccttgggcacatggtacgacctgcagacggtgccaatagaggcccttggcagcctcccaccccacctgcagctccccgtgagccagccatccacagcgaggggcagtgggtgacgctgccggcccccctggacaccatcaacgtccacctccgggctgggtacatcatccccctgcagggccctggcctcacaaccacagagtcccgccagcagcccatggccctggctgtggccctgaccaagggtggagaggcccgaggggagctgttctgggacgatggagagagcctggaagtgctggagcgaggggcctacacacaggtcatcttcctggccaggaataacacgatcgtgaatgagctggtacgtgtgaccagtgagggagctggcctgcagctgcagaaggtgactgtcctgggcgtggccacggcgccccagcaggtcctctccaacggtgtcccgtctccaacttcacctacagccccgacaccaaggtcctggacatctgtgtctcgctgttgatgggagagcagtttctcgtcagctggtgttagtctagagcttgctagcggccgcConstruct 1758

The GILTΔ2-7Δ34-39-GAA70-952 cassette below was cloned using the Asp718and NotI sites of the cassette and vector pCEP4 to producepCEP-GILTΔ2-7Δ34-39-GAA70-952 (Plasmid 1758). Restriction sites forcloning are in lowercase bold. The spacer amino acid sequence Gly, Ala,Pro (underlined sequence) separate the GAA gene and GILTΔ2-7Δ34-39 tag(upper case sequence). The spacer and tag are placed upstream of GAAresidue Ala70. The GILTΔ2-7Δ34-39 cassette contains a deletion of aminoacid residues 34-39 (Arg-Val-Ser-Arg-Arg-Ser) from the human IGF-IIsequence.

(SEQ ID NO: 22) ggtaccagctgctagcaagctaattcacaccaATGGGAATCCCAATGGGGAAGTCGATGCTGGTGCTTCTCACCTTCTTGGCCTTCGCCTCGTGCTGCATTGCTGCTCTGTGCGGCGGGGAGCTGGTGGACACCCTCCAGTTCGTCTGTGGGGACCGCGGCTTCTACTTCAGCAGGCCCGCAAGCCGTGGCATCGTTGAGGAGTGCTGTTTCCGCAGCTGTGACCTGGCCCTCCTGGAGACGTACTGTGCTACCCCCGCCAAGTCCGAGGGCGCGCCGgcacaccccggccgtcccagagcagtgcccacacagtgcgacgtcccccccaacagccgcttcgattgcgcccctgacaaggccatcacccaggaacagtgcgaggcccgcggctgctgctacatccctgcaaagcaggggctgcagggagcccagatggggcagccctggtgcttcttcccacccagctaccccagctacaagctggagaacctgagctcctctgaaatgggctacacggccaccctgacccgtaccacccccaccttcttccccaaggacatcctgaccctgcggctggacgtgatgatggagactgagaaccgcctccacttcacgatcaaagatccagctaacaggcgctacgaggtgcccttggagaccccgcgtgtccacagccgggcaccgtccccactctacagcgtggagttctctgaggagcccttcggggtgatcgtgcaccggcagctggacggccgcgtgctgctgaacacgacggtggcgcccctgactttgcggaccagaccttcagctgtccacctcgctgccctcgcagtatatcacaggcctcgccgagcacctcagtcccctgatgctcagcaccagctggaccaggatcaccctgtggaaccgggaccttgcgcccacgcccggtgcgaacctctacgggtctcaccattctacctggcgctggaggacggcgggtcggcacacggggtgttcctgctaaacagcaatgccatggatgtggtcctgcagccgagccctgccatagctggaggtcgacaggtgggatcctggatgtctacatcttcctgggcccagagcccaagagcgtggtgcagcagtacctggacgttgtgggatacccgttcatgccgccatactggggcctgggcttccacctgtgccgctggggctactcctccaccgctatcacccgccaggtggtggagaacatgaccagggcccacttccccctggacgtccaatggaacgacctggactacatggactcccggagggacttcacgttcaacaaggatggatccgggacttcccggccatggtgcaggagctgcaccagggcggccggcgctacatgatgatcgtggatcctgccatcagcagctcgggccctgccgggagctacaggccctacgacgagggtctgcggaggggggttttcatcaccaacgagaccggccagccgctgattgggaaggtatggcccgggtccactgccttccccgacttcaccaaccccacagccctggcctggtgggaggacatggtggctgagttccatgaccaggtgccatcgacggcatgtggattgacatgaacgagccttccaacttcatcaggggctctgaggacggctgccccaacaatgagctggagaacccaccctacgtgcctggggtggttggggggaccctccaggcggcaaccatctgtgcctccagccaccagtactctccacacactacaacctgcacaacctctacggcctgaccgaagccatcgcctcccacagggcgctggtgaaggctcgggggacacgcccatttgtgatctcccgctcgacctttgctggccacggccgatacgccggccactggacgggggacgtgtggagctcctgggagcagctcgcctcctccgtgccagaaatcctgcagtttaacctgctgggggtgcctctggtcggggccgacgtctgcggcttcctgggcaacacctcagaggagctgtgtgtgcgctggacccagctgggggccttctaccccttcatgcggaaccacaacagcctgctcagtctgccccaggagccgtacagatcagcgagccggcccagcaggccatgaggaaggccctcaccctgcgctacgcactcctcccccacctctacacgctgttccaccaggcccacgtcgcgggggagaccgtggcccggcccctcttcctggagttccccaaggactctagcacctggactgtggaccaccagctcctgtggggggaggccctgctcatcaccccagtgctccaggccgggaaggccgaagtgactggctacttccccttgggcacatggtacgacctgcagacggtgccaatagaggccatggcagcctcccacccccacctgcagctccccgtgagccagccatccacagcgaggggcagtgggtgacgctgccggcccccctggacaccatcaacgtccacctccgggctgggtacatcatccccctgcagggccctggcctcacaaccacagagtcccgccagcagcccatggccctggctgtggccctgaccaagggtggagaggcccgaggggagctgttctgggacgatggagagagcctggaagtgctggagcgaggggcctacacacaggtcatcttcctggccaggaataacacgatcgtgaatgagctggtacgtgtgaccagtgagggagctggcctgcagctgcagaaggtgactgtcctgggcgtggccacggcgccccagcaggtcctctccaacggtgtccctgtctccaacttcacctacagccccgacaccaaggtcctggacatctgtgtctcgctgttgatgggagagcagtttctcgtcagctggtgttagtctagagcttgctagcggccgcConstruct 1750

The GILTΔ2-7Δ35-39-GAA70-952 cassette below was cloned using the Asp718and NotI sites of the cassette and vector pCEP4 to producepCEP-GILTΔ2-7Δ35-39-GAA70-952 (Plasmid 1750). Restriction sites forcloning are in lowercase bold. The spacer amino acid sequence Gly, Ala,Pro (underlined sequence) separate the GAA gene and GILTΔ2-7Δ35-39 tag(upper case sequence). The spacer and tag are placed upstream of GAAresidue Ala70. The GILTΔ2-7Δ35-39 cassette contains a deletion of aminoacid residues 35-39 (Val-Ser-Arg-Arg-Ser) from the human IGF-IIsequence.

(SEQ ID NO: 23) ggtaccagagctagcaagctaattcacaccaATGGGAATCCCAATGGGGAAGTCGATGCTGGTGCTTCTCACCTTCTTGGCCTTCGCCTCGTGCTGCATTGCTGCTCTGTGCGGCGGGGAGCTGGTGGACACCCTCCAGTTCGTCTGTGGGGACCGCGGCTTCTACTTCAGCAGGCCCGCAAGCCGTCGTGGCATCGTTGAGGAGTGCTGTTTCCGCAGCTGTGACCTGGCCCTCCTGGAGACGTACTGTGCTACCCCCGCCAAGTCCGAGGGCGCGCCGgcacaccccggccgtcccagagcagtgcccacacagtgcgacgtcccccccaacagccgatcgattgcgcccagacaaggccatcacccaggaacagtgcgaggcccgcggctgctgctacatccctgcaaagcaggggctgcagggagcccagatggggcagccctggtgcttatcccacccagctaccccagctacaagctggagaacctgagctcctagaaatgggctacacggccaccctgacccgtaccacccccaccttatccccaaggacatcctgaccctgcggctggacgtgatgatggagactgagaaccgcctccacttcacgatcaaagatccagctaacaggcgctacgaggtgcccttggagaccccgcgtgtccacagccgggcaccgtccccactctacagcgtggagactctgaggagcccttcggggtgatcgtgcaccggcagctggacggccgcgtgctgctgaacacgacggtggcgcccctgttattgcggaccagttccttcagagtccacctcgctgccctcgcagtatatcacaggcctcgccgagcacctcagtcccctgatgacagcaccagaggaccaggatcaccctgtggaaccgggaccttgcgcccacgcccggtgcgaacctctacgggtctcaccattctacctggcgctggaggacggcgggtcggcacacggggtgacctgctaaacagcaatgccatggatgtggtcctgcagccgagccctgcccttagctggaggtcgacaggtgggatcctggatgtctacatatcctgggcccagagcccaagagcgtggtgcagcagtacctggacgttgtgggatacccgttcatgccgccatactggggcctgggcttccacctgtgccgctggggctactcctccaccgctatcacccgccaggtggtggagaacatgaccagggcccacttccccctggacgtccaatggaacgacctggactacatggactcccggagggacttcacgttcaacaaggatggatccgggacttcccggccatggtgcaggagagcaccagggcggccggcgctacatgatgatcgtggatcctgccatcagcagctcgggccctgccgggagctacaggccctacgacgagggtctgcggaggggggttttcatcaccaacgagaccggccagccgctgattgggaaggtatggcccgggtccactgccttccccgacttcaccaaccccacagccctggcctggtgggaggacatggtggctgagttccatgaccaggtgcccttcgacggcatgtggattgacatgaacgagccttccaacttcatcaggggctctgaggacggctgccccaacaatgagctggagaacccaccctacgtgcctggggtggttggggggaccaccaggcggcaaccatagtgcctccagccaccagtttctaccacacactacaacctgcacaacctctacggcctgaccgaagccatcgcctcccacagggcgctggtgaaggctcgggggacacgcccatttgtgatctcccgctcgacctagctggccacggccgatacgccggccactggacgggggacgtgtggagctcctgggagcagctcgcctcctccgtgccagaaatcctgcagtttaacctgctgggggtgcctaggtcggggccgacgtctgcggcttcctgggcaacacctcagaggagctgtgtgtgcgctggacccagagggggccttctacccatcatgcggaaccacaacagcctgctcagtctgccccaggagccgtacagcttcagcgagccggcccagcaggccatgaggaaggccctcaccctgcgctacgcactcctcccccacctctacacgctgttccaccaggcccacgtcgcgggggagaccgtggcccggcccctcttcctggagttccccaaggactctagcacctggactgtggaccaccagtcctgtggggggaggccagctcatcaccccagtgaccaggccgggaaggccgaagtgactggctacttccccttgggcacatggtacgacctgcagacggtgccaatagaggcccttggcagcctcccacccccacctgcagctccccgtgagccagccatccacagcgaggggcagtgggtgacgctgccggcatccctggacaccatcaacgtccacctccgggctgggtacatcatccccctgcagggccctggcctcacaaccacagagtcccgccagcagcccatggccaggctgtggccctgaccaagggtggagaggcccgaggggagctgttctgggacgatggagagagcctggaagtgctggagcgaggggcctacacacaggtcatcttcctggccaggaataacacgatcgtgaatgagaggtacgtgtgaccagtgagggagctggcctgcagctgcagaaggtgactgtcctgggcgtggccacggcgccccagcaggtcctctccaacggtgtccctgtctccaacttcacctacagcccgacaccaaggtcctggacatctgtgtctcgagttgatgggagagcagtttctcgtcagctggtgttagtctagagcttgctagcggccgcConstruct 1748

The GILTΔ2-7Δ36-39-GAA70-952 cassette below was cloned using the Asp718and NotI sites of the cassette and vector pCEP4 to producepCEP-GILTΔ2-7Δ36-39-GAA70-952 (Plasmid 1748). Restriction sites forcloning are in lowercase bold. The spacer amino acid sequence Gly, Ala,Pro (underlined sequence) separate the GAA gene and GILTΔ2-7Δ36-39 tag(upper case sequence). The spacer and tag are placed upstream of GAAresidue Ala70. The GILTΔ2-7Δ36-39 cassette contains a deletion of aminoacid residues 36-39 (Ser-Arg-Arg-Ser) from the human IGF-II sequence.

(SEQ ID NO: 24) ggtaccagctgctagcaagctaattcacaccaATGGGAATCCCAATGGGGAAGTCGATGCTGGTGCTTCTCACCTTCTTGGCCTTCGCCTCGTGCTGCATTGCTGCTCTGTGCGGCGGGGAGCTGGTGGACACCCTCCAGTTCGTCTGTGGGGACCGCGGCTTCTACTTCAGCAGGCCCGCAAGCCGTGTGCGTGGCATCGTTGAGGAGTGCTGTTTCCGCAGCTGTGACCTGGCCCTCCTGGAGACGTACTGTGCTACCCCCGCCAAGTCCGAGGGCGCGCCGgcacaccccggccgtcccagagcagtgcccacacagtgcgacgtcccccccaacagccgatcgattgcgcccctgacaaggccatcacccaggaacagtgcgaggcccgcggctgctgctacatccctgcaaagcaggggctgcagggagcccagatggggcagccctggtgatcttcccacccagctaccccagctacaagaggagaacctgagctcactgaaatgggctacacggccaccagacccgtaccacccccaccttcttccccaaggacatcctgaccctgcggaggacgtgatgatggagactgagaaccgcctccacttcacgatcaaagatccagctaacaggcgctacgaggtgcccttggagaccccgcgtgtccacagccgggcaccgtccccactctacagcgtggagttactgaggagcccttcggggtgatcgtgcaccggcagctggacggccgcgtgagctgaacacgacggtggcgcccctgttattgcggaccagttcatcagagtccacctcgctgccctcgcagtatatcacaggcctcgccgagcacctcagtcccctgatgctcagcaccagctggaccaggatcaccctgtggaaccgggaccttgcgcccacgcccggtgcgaacctctacgggtctcaccctttctacctggcgctggaggacggcgggtcggcacacggggtgttcctgctaaacagcaatgccatggatgtggtcctgcagccgagccagcccttagctggaggtcgacaggtgggatcctggatgtctacatcttcctgggcccagagcccaagagcgtggtgcagcagtacctggacgagtgggatacccgttcatgccgccatactggggcctgggcttccacctgtgccgctggggctactcctccaccgctatcacccgccaggtggtggagaacatgaccagggcccacttccccctggacgtccaatggaacgacctggactacatggactcccggagggacttcacgttcaacaaggatggcttccgggacttcccggccatggtgcaggagctgcaccagggcggccggcgctacatgatgatcgtggatcctgccatcagcagacgggccagccgggagctacaggccctacgacgagggtctgcggaggggggattcatcaccaacgagaccggccagccgctgattgggaaggtatggcccgggtccactgccttccccgacttcaccaaccccacagccctggcctggtgggaggacatggtggctgagttccatgaccaggtgcccttcgacggcatgtggattgacatgaacgagccttccaacttcatcaggggctctgaggacggctgccccaacaatgagctggagaacccaccctacgtgcctggggtggttggggggaccctccaggcggcaaccatagtgcctccagccaccagtttactccacacactacaacctgcacaacctctacggcctgaccgaagccatcgcctcccacagggcgctggtgaaggctcgggggacacgcccatttgtgatctcccgctcgacctttgctggccacggccgatacgccggccactggacgggggacgtgtggagctcctgggagcagctcgcctcctccgtgccagaaatcctgcagtttaacctgctgggggtgcctctggtcggggccgacgtctgcggcttcctgggcaacacctcagaggagagtgtgtgcgctggacccagctgggggccttctacccatcatgcggaaccacaacagcctgctcagtagccccaggagccgtacagatcagcgagccggcccagcaggccatgaggaaggccacaccctgcgctacgcactcctcccccacctctacacgctgttccaccaggcccacgtcgcgggggagaccgtggcccggcccctcttcctggagttccccaaggactctagcacctggactgtggaccaccagacctgtggggggaggccctgctcatcaccccaggctccaggccgggaaggccgaagtgactggctacttccccttgggcacatggtacgacctgcagacggtgccaatagaggccatggcagcctcccacccccacctgcagctccccgtgagccagccatccacagcgaggggcagtgggtgacgctgccggcccccctggacaccatcaacgtccacctccgggagggtacatcatccccctgcagggccctggcctcacaaccacagagtcccgccagcagcccatggccctggctgtggccctgaccaagggtggagaggcccgaggggagagttctgggacgatggagagagcctggaagtgaggagcgaggggcctacacacaggtcatcttcctggccaggaataacacgatcgtgaatgagctggtacgtgtgaccagtgagggagctggcctgcagctgcagaaggtgactgtcctgggcgtggccacggcgccccagcaggtcctctccaacggtgtccctgtctccaacttcacctacagccccgacaccaaggtcctggacatctgtgtctcgctgttgatgggagagcagtttctcgtcagctggtgttagtctagagcttgctagcggccgcConstruct 1751

The GILTΔ2-7Δ29-40-GAA70-952 cassette below was cloned using the Asp718and NotI sites of the cassette and vector pCEP4 to producepCEP-GILTΔ2-7Δ29-40-GAA70-952 (Plasmid 1751). Restriction sites forcloning are in lowercase bold. The spacer amino acid sequence Gly, Ala,Pro (underlined sequence) separate the GAA gene and GILTΔ2-7Δ29-40 tag(upper case sequence). The spacer and tag are placed upstream of GAAresidue Ala70. The GILTΔ2-7Δ29-40 cassette contains a deletion of aminoacid residues 29-40 (Ser-Arg-Pro-Ala-Ser-Arg-Val-Ser-Arg-Arg-Ser-Arg)from the human IGF-II sequence.

(SEQ ID NO: 25) ggtaccagctgctagcaagctaattcacaccaATGGGAATCCCAATGGGGAAGTCGATGCTGGTGCTTCTCACCTTCTTGGCCTTCGCCTCGTGCTGCATTGCTGCTCTGTGCGGCGGGGAGCTGGTGGACACCCTCCAGTTCGTCTGTGGGGACCGCGGCTTCTACTTCGGCATCGTTGAGGAGTGCTGTTTCCGCAGCTGTGACCTGGCCCTCCTGGAGACGTACTGTGCTACCCCCGCCAAGTCCGAGGGCGCGCCGgcacaccccggccgtcccagagcagtgcccacacagtgcgacgtcccccccaacagccgcttcgattgcgcccctgacaaggccatcacccaggaacagtgcgaggcatgcggctgctgctacatccctgcaaagcaggggctgcagggagcccagatggggcagccaggtgcttcttcccacccagctaccccagctacaagctggagaacctgagctcctctgaaatgggctacacggccaccctgacccgtaccacccccaccacttccccaaggacatcctgaccagcggctggacgtgatgatggagactgagaaccgcctccacttcacgatcaaagatccagctaacaggcgctacgaggtgcccttggagaccccgcgtgtccacagccgggcaccgtccccactctacagcgtggagttctctgaggagcccttcggggtgatcgtgcaccggcagctggacggccgcgtgctgctgaacacgacggtggcgcccctgttctttgcggaccagttccttcagagtccacctcgctgccctcgcagtatatcacaggcctcgccgagcacctcagtcccctgatgctcagcaccagctggaccaggatcaccctgtggaaccgggaccttgcgcccacgcccggtgcgaacctctacgggtctcaccctttctacctggcgctggaggacggcgggtcggcacacggggtgttcctgctaaacagcaatgccatggatgtggtcctgcagccgagccctgcccttagctggaggtcgacaggtgggatcctggatgtctacatcttcctgggcccagagcccaagagcgtggtgcagcagtacctggacgttgtgggatacccgttcatgccgccatactggggcctgggcttccacctgtgccgctggggctactcctccaccgctatcacccgccaggtggtggagaacatgaccagggcccacttccccctggacgtccaatggaacgacctggactacatggactcccggagggacttcacgttcaacaaggatggcttccgggacttcccggccatggtgcaggagctgcaccagggcggccggcgctacatgatgatcgtggatcctgccatcagcagctcgggccctgccgggagctacaggccctacgacgagggtagcggaggggggttttcatcaccaacgagaccggccagccgctgattgggaaggtatggcccgggtccactgccttccccgacttcaccaaccccacagccctggcctggtgggaggacatggtggctgagttccatgaccaggtgcccttcgacggcatgtggattgacatgaacgagcatccaacttcatcaggggctctgaggacggctgccccaacaatgagctggagaacccaccctacgtgcctggggtggttggggggaccaccaggcggcaaccatctgtgcctccagccaccagtttctaccacacactacaacctgcacaacctctacggcctgaccgaagccatcgcctcccacagggcgctggtgaaggctcgggggacacgcccatttgtgatctcccgctcgacattgctggccacggccgatacgccggccactggacgggggacgtgtggagctcctgggagcagctcgcctcctccgtgccagaaatcctgcagtttaacctgctgggggtgcctaggtcggggccgacgtctgcggcttcctgggcaacacctcagaggagctgtgtgtgcgctggacccagctgggggccttctaccccttcatgcggaaccacaacagcctgacagtagccccaggagccgtacagcttcagcgagccggcccagcaggccatgaggaaggccctcaccctgcgctacgcactcctcccccacctctacacgctgttccaccaggcccacgtcgcgggggagaccgtggcccggcccctatcctggagttccccaaggactctagcacctggactgtggaccaccagctcctgtggggggaggccagacatcaccccagtgctccaggccgggaaggccgaagtgactggctacttcccatgggcacatggtacgacctgcagacggtgccaatagaggcccttggcagcctcccacccccacctgcagctccccgtgagccagccatccacagcgaggggcagtgggtgacgctgccggcccccctggacaccatcaacgtccacctccgggctgggtacatcatccccctgcagggccctggcctcacaaccacagagtcccgccagcagcccatggccctggctgtggccagaccaagggtggagaggcccgaggggagctgttctgggacgatggagagagcctggaagtgctggagcgaggggcctacacacaggtcatatcctggccaggaataacacgatcgtgaatgagctggtacgtgtgaccagtgagggagaggcctgcagctgcagaaggtgactgtcctgggcgtggccacggcgccccagcaggtcctaccaacggtgtccctgtctccaacttcacctacagccccgacaccaaggtcctggacatctgtgtacgctgttgatgggagagcagtttctcgtcagctggtgttagtctagagct tgctagcggccgcConstruct 1752

The GILTΔ2-7Δ30-40-GAA70-952 cassette below was cloned using the Asp718and NotI sites of the cassette and vector pCEP4 to producepCEP-GILTΔ2-7Δ30-40-GAA70-952 (Plasmid 1752). Restriction sites forcloning are in lowercase bold. The spacer amino acid sequence Gly, Ala,Pro (underlined sequence) separate the GAA gene and GILTΔ2-7Δ30-40 tag(upper case sequence). The spacer and tag are placed upstream of GAAresidue Ala70. The GILTΔ2-7Δ30-40 cassette contains a deletion of aminoacid residues 30-40 (Arg-Pro-Ala-Ser-Arg-Val-Ser-Arg-Arg-Ser-Arg) fromthe human IGF-II sequence.

(SEQ ID NO: 26) ggtaccagctgctagcaagctaattcacaccaATGGGAATCCCAATGGGGAAGTCGATGCTGGTGCTTCTCACCTTCTTGGCCTTCGCCTCGTGCTGCATTGCTGCTCTGTGCGGCGGGGAGCTGGTGGACACCCTCCAGTTCGTCTGTGGGGACCGCGGCTTCTACTTCAGCGGCATCGTTGAGGAGTGCTGTTTCCGCAGCTGTGACCTGGCCCTCCTGGAGACGTACTGTGCTACCCCCGCCAAGTCCGAGGGCGCGCCGgcacaccccggccgtcccagagcagtgcccacacagtgcgacgtcccccccaacagccgcttcgattgcgcccctgacaaggccatcacccaggaacagtgcgaggcccgcggctgctgctacatccctgcaaagcaggggctgcagggagcccagatggggcagccctggtgcttcttcccacccagctaccccagctacaagctggagaacctgagctcctctgaaatgggctacacggccaccctgacccgtaccacccccaccttcctccccaaggacatcctgaccctgcggctggacgtgatgatggagactgagaaccgcctccacttcacgatcaagatccagctaacaggcgctacgaggtgcccttggagaccccgcgtgtccacagccgggcaccgtccccactcacagcgtggagttctctgaggagcccttcggggtgatcgtgcaccggcagctggacggccgcgtgctgctgaacacgacggtggcgcccctgttctttgcggaccagttccttcagctgtccacctcgctgccctcgcagtatatcacaggcctcgccgagcacctcagtccctgatgctcagcaccagctggaccaggatcacctgtggaaccgggaccttgcgcccacgcccggtgcgaacctctacgggtctcaccctttctacctggcgctggaggtcgacaggtggatcctggatgtctacatcttcctgggcccagagcccaagagcgtggtgcagcagtacctggacgttgtgggatacccgttcatgccgccatactggggcctgggcttccacctgtgccgctggggctactcctccaccgctatcacccgccaggtggtggagaacatgaccagggcccacttccccctggacgtccaatggaacgacctggactacatggactcccggagggacttcacgttcaacaaggatggcttccgggacttcccggccatggtgcaggagctgcaccagggcggccggcgctacatgatgatcgtggatcctgccatcagcagctcgggccctgccgggagctacaggccctacgacgagggtctgcggaggggggtttttcatcaccaacgagaccggccagccgctgattgggaaggtatggcccgggtccactgccttccccgacttcaccaaccccacagccctggcctggtgggaggacatggtggctgagttccatgaccaggtgcccttcgacggcatgtggattgacatgaacgagcctccaacttcatcaggggctctgaggacggctgccccaacaatgagctggagaacccaccctacgtgcctggggtggtggggggaccctccaggcggcaaccatctgtgcctccagccaccagtttctctccacacactacaacctgcacaacctctacggcctgaccgaagccatcgcctcccacagggcgctggtgaaggctcgggggacacgcccatttgtgatctcccgctcgacctttgctggccacggccgatacgccggccactggacgggggacgtgtggagctcctgggagcagctcgcctcctccgtgccagaaatcctgcagtttaacctgctgggggtgcctctggtcggggccgacgtctgcggcttcctgggcaacacctcagaggagctgtgtgtgcgctggacccagctgggggccttctaccccttcatgcggaaccacaacagcctgctcagtctgccccaggagccgtacagcttcagcgagccggcccagcaggccatgaggaaggccctcaccctgcgctacgcactcctcccccacctctacacgctgttccaggcccacgtcgcgggggagaccgtggcccggcccctcttcctggagttccccaaggactctagcacctggactgtggaccaccagctcctgtggggggaggccctgctcatcaccccagtgctccaggccgggaaggccgaagtgactggctacttccccttgggcacatggtacgacctgcagacggtgccaatagaggcccttggcagcctcccacccccacctgcagctccccgtgagccagcctccacgcgaggggcagtgggtgacgctgccggccccctggacaccatcaacgtccacctccgggctgggtacatcatccccctgcagggccctggcctcacaaccacagagtcccgccagcagcccatggccctggctgtggccctgaccaagggtggagaggcccgaggggagctgttctgggacgatggagagagcctggaagtgctggagcgaggggcctacacaggtcatgttcctggccaggaataacacgatcgtgaatgagctggtacgtgtgaccagtgagggagctggcctgcagctgcagaaggtgactgtcctgggcgtggccacggcgccccagaggtcctctccaacggtgtccctgtctccaacttcacctacagccccgacaccaaggtcctggacatctgtgtctcgctgttgatgggagagcagtttctcgtcagctggtgttagtctagagcttgctagcggccgcConstruct 1753

The GILTΔ2-7Δ31-40-GAA70-952 cassette below was cloned using the Asp718and NotI sites of the cassette and vector pCEP4 to producepCEP-GILTΔ2-7Δ31-40-GAA70-952 (Plasmid 1753). Restriction sites forcloning are in lowercase bold. The spacer amino acid sequence Gly, Ala,Pro (underlined sequence) separate the GAA gene and GILTΔ2-7Δ31-40 tag(upper case sequence). The spacer and tag are placed upstream of GAAresidue Ala70. The GILTΔ2-7Δ31-40 cassette contains a deletion of aminoacid residues 31-40 (Pro-Ala-Ser-Arg-Val-Ser-Arg-Arg-Ser-Arg) from thehuman IGF-II sequence.

(SEQ ID NO: 27) ggtaccagctgctagcaagctaattcacaccaATGGGAATCCCAATGGGGAAGTCGATGCTGGTGCTTCTCACCATTCTTGGCCTTCGCCTCGTGCTGCATTGCTGCTCTGTGCGGCGGGGAGCTGGTGGACACCCTCCAGTTCGTCTGTGGGGACCGCGGCTTCTACTTCAGCAGGGGCATCGTTGAGGAGTGCTGTTTCCGCAGCTGTGACCTGGCCCTCCTGGAGACGTACTGTGCTACCCCCGCCAAGTCCGAGGGCGCGCCGgcacaccccggccgtgggagagcagtgcccacacagtgcgacgtcccccccaacagccgcttcgattgcgccctgacaaggccatcacccaggaacaggacatcctgaccctgcggctggacgtgatgatggagactggggctgcagggagcccagatggggcagccctggtgcttcttcccacccagctaccccagctacaagctggagaacctgagctcctctgaaatgggctacacggccaccctgacccgtaccacccccaccttcttccccaaggacatcctgaccctgcggctggacgtgatgatggagactgagaaccgcctccacttcacgatcaaagatccagctaacaggcgctacgaggtgcccttggagaccccgcgtgtccacagccgggcaccgtccccactctacagcgtggagttctctgaggagcccttcggggtgatcgtgcaccggcagctggacggccgcgtgctgctgaacacgacggtggcgcccctgttctttgcggaccagttccttcagctgtccacctcgctgccctcgcagtatatcacaggcctcgccgagcacctcagtcccctgatgctcagcaccagctggaccaggatcaccctgtggaaccgggaccttgcgcccacgcccggtgcgaacctctacgggtctcaccctttctacctggcgctggaggacggcgggtcggcacacggggtgttcctgctaaacagcaatgccatggatgtggtcctgcagccgagccctgcccttagctggaggtcgacaggtgggatcctggatgtctacatcttcctgggcccagagcccaagagcgtggtgcagcagtacctggacgttgtgggatacccgttcatgccgccatactggggcctgggcttccacctgtgccgctggggctactcctccaccgctatcacccgccaggtggtggagaacatgaccagggcccacttccccctggacgtccaatggaacgacctggactacatggactcccggagggacttcacgttcaacaaggatggcttccgggacttcccggccatggtgcaggagctgcaccagggcggccggcgctacatgatgatcgtggatcctgccatcagcagctcgggccctgccgggagctacaggccctacgacgagggtctgcggaggggggttttcatcaccaacgagaccggccagccgctgattgggaaggtatggcccgggtccactgccttccccgacttcaccaacccacagccctggcctggtgggaggacatggtggctgagttccatgaccaggtgcccttcgacggcatgtggattgacatgaacgagccttccaacttcatcaggggctctgaggacggctgccccaacaatgagctggagaacccaccctacgtgcctggggtggttggggggaccctccaggcggcaaccatctgtgcctccagccaccagtttctctccacacactacaacctgcacaacctctacggctgaccgaagccatcgcctcccacagggcgctggtgaaggctcggggacacgcccatttgtgatctcccgctcgacctttgctggccacggccgatacgccggccggccactggacgggggacgtgtggagctcctgggagcagctcgcctcctccgtgccagaaatcctgcagtttaacctgctgggggtgcctctggtcggggccgacgtctgcggcttcctgggcaacacctcagaggagctgtgtgtgcgctggacccagctgggggccttctaccccttcatgcggaaccacaacagcctgctcagtctgccccaggagccgtcagcttcagcgagccggcccagcaggccatgaggaaggccctcaccctcgcgtacgcactcctcccccacctctacacgctgttccaccaggcccacgtcgcgggggagaccgtggcccggcccctcttcctggagttccccaaggactctagcacctggactgtggaccaccagctcctgtggggggaggcctgctcatcaccccagtgctccaggccgggaaggccgaagtgactggctacttccccttgggcacatggtacgacctgcagacggtgccaatagaggcccttggcagcctcccacccccacctgcagctccccgtgagccagccatccacagcgaggggcagtgggtgacgctgccggcccccctggacaccatcaacgtccacctccgggctgggtacatcatccccctgcagggccctggcctcacaaccacagagtcccgccagcagcccatggccctggctgtggccctgaccaagggtggagaggcccgaggggagctgttctgggacgatggagagagcctggagtgctggagcgaggggcctacacaggtcatcttcctggccaggaataacacgatcgtgaatgagctggtacgtgtgaccagtgagggagctggcctgcagctgcagaaggtgactgtcctgggcgtggccacggcgccccagcaggtcctctccacggtgtccctgtctccaacttcacctacagccccgacaccaaggtcctggacatctgtgtctcgctggttgatgggagagcagtttctctgtcagctggtgttagtctagagcttgctagcggccgcConstruct 1754

The GILTΔ2-7Δ32-40-GAA70-952 cassette below was cloned using the Asp718and NotI sites of the cassette and vector pCEP4 to producepCEP-GILTΔ2-7Δ32-40-GAA70-952 (Plasmid 1754). Restriction sites forcloning are in lowercase bold. The spacer amino acid sequence Gly, Ala,Pro (underlined sequence) separate the GAA gene and GILTΔ2-7Δ32-40 tag(upper case sequence). The spacer and tag are placed upstream of GAAresidue Ala70. The GILTΔ2-7Δ32-40 cassette contains a deletion of aminoacid residues 32-40 (Ala-Ser-Arg-Val-Ser-Arg-Arg-Ser-Arg) from the humanIGF-II sequence.

(SEQ ID NO: 28) ggtaccagctgctagcaagctaattcacaccaATGGGAATCCCAATGGGGAAGTCGATGCTGGTGCTTCTCACCTTCTTGGCCTTCGCCTCGTGCTGCATTGCTGCTCTGTGCGGCGGGGAGCTGGTGGACACCCTCCAGTTCGTCTGTGGGGACCGCGGCTTCTACTTCAGCAGGCCCGGCATCGTTGAGGAGTGCTGTTCCGCAGCTGTGACCTGGCCCTCCTGGAGACGTACTGTGCTACCCCCGCCAAGTCCGAGGGCGCGCCGgcacaccccggccgtcccagagcagtgcccacacagtgcgacgtcccccccaacagccgcttcgattgcgcccctgacaaggccatcacccaggaacagtgcgaggcccgcggctgctgctacatccctgcaaagcaggggctgcagggagcccagatggggcagccctggtgatcttcccacccagctaccccagctacaagctggagaacctgagctcctctgaaatgggctacacggccaccctgacccgtaccacccccaccttcttccccaaggacatcctgaccctgcggctggacgtgatgatggagactgagaaccgcctccacttcacgatcaaagatccagctaacaggcgctacgaggtgcccttggagaccccgcgtgtccacagccgggcaccgtccccactctacagcgtggagttctctgaggagccatcggggtgatcgtgcaccggcagctggacggccgcgtgctgctgaacacgacggtggcgcccctgttctttgcggaccagttccttcagctgtccacctcgctgccctcgcagtatatcacaggcctcgccgagcacctcagtcccctgatgctcagcaccagctggaccaggatcaccctgtggaaccgggaccttgcgcccacgcccggtgcgaacctctacgggtctcaccattctacctggcgctggaggacggcgggtcggcacacggggtgttcctgctaaacagcaatgccatggatgtggtcctgcagccgagccctgcccttagctggaggtcgacaggtgggatcctggatgtctacatcttcctgggcccagagcccaagagcgtggtgcagcagtacctggacgttgtgggatacccgttcatgccgccatactggggcctgggcttccacctgtgccgctggggctactcctccaccgctatcacccgccaggtggtggagaacatgaccagggcccacttccccctggacgtccaatggaacgacctggactacatggactcccggagggacttcacgttcaacaaggatggcttccgggacttcccggccatggtgcaggagctgcaccagggcggccggcgctacatgatgatcgtggatcctgccatcagcagctcgggccctgccgggagctacaggccctacgacgagggtctgcggaggggggttttcatcaccaacgagaccggccagccgctgattgggaaggtatggcccgggtccactgccttccccgacttcaccaaccccacagccctggcctggtgggaggacatggtggctgagttccatgaccaggtgcccttcgacggcatgtggattgacatgaacgagccttccaacttcatcaggggctctgaggacggctgccccaacaatgagctggagaacccaccctacgtgcctggggtggttggggggaccctccaggcggcaaccatctgtgcctccagccaccagtttctctccacacactacaacctgcacaacctctacggcctgaccgaagccatcgcctcccacagggcgctggtgaaggctcgggggacacgcccatttgtgatctcccgctcgacctttgctggccacggccgatacgccggccactggacgggggacgtgtggagctcctgggagcagctcgcctcctccgtgccagaaatcctgcagtttaacctgctgggggtgcctctggtcggggccgacgtctgcggcttcctgggcaacacctcagaggagagtgtgtgcgctggacccagctgggggccttctacccatcatgcggaaccacaacagcctgctcagtctgccccaggagccgtacagcttcagcgagccggcccagcaggccatgaggaaggccctcaccctgcgctacgcactcctcccccacctctacacgctgttccaccaggcccacgtcgcgggggagaccgtggcccggcccctcttcctggagttccccaaggactctagcacctggactgtggaccaccagctcctgtggggggaggccctgctcatcaccccagtgctccaggccgggaaggccgaagtgactggctacttccccttgggcacatggtacgacctgcagacggtgccaatagaggccatggcagcctcccacccccacctgcagctccccgtgagccagccatccacagcgaggggcagtgggtgacgctgccggcccccctggacaccatcaacgtccacctccgggctgggtacatcatccccctgcagggccctggcctcacaaccacagagtcccgccagcagcccatggccctggctgtggccctgaccaagggtggagaggcccgaggggagctgttctgggacgatggagagagcctggaagtgctggagcgaggggcctacacacaggtcatcttcctggccaggaataacacgatcgtgaatgagctggtacgtgtgaccagtgagggagctggcctgcagctgcagaaggtgactgtcctgggcgtggccacggcgccccagcaggtcctctccaacggtgtccctgtctccaacttcacctacagccccgacaccaaggtcctggacatctgtgtctcgctgttgatgggagagcagtttctcgtcagctggtgttagtctagagcttgctagcggccgcConstruct 1755

The GILTΔ2-7Δ33-40-GAA70-952 cassette below was cloned using the Asp718and NotI sites of the cassette and vector pCEP4 to producepCEP-GILTΔ2-7Δ33-40-GAA70-952 (Plasmid 1755). Restriction sites forcloning are in lowercase bold. The spacer amino acid sequence Gly, Ala,Pro (underlined sequence) separate the GAA gene and GILTΔ2-7Δ33-40 tag(upper case sequence). The spacer and tag are placed upstream of GAAresidue Ala70. The GILTΔ2-7Δ33-40 cassette contains a deletion of aminoacid residues 33-40 (Ser-Arg-Val-Ser-Arg-Arg-Ser-Arg) from the humanIGF-II sequence.

(SEQ ID NO: 29) ggtaccagctgctagcaagctaattcacaccaATGGGAATCCCAATGGGGAAGTCGATGCTGGTGCTTCTCACCTTCTTGGCCTTCGCCTCGTGCTGCATTGCTGCTCTGTGCGGCGGGGAGCTGGTGGACACCCTCCAGTTCGTCTGTGGGGACCGCGGCTTCTACTTCAGCAGGCCCGCAGGCATCGTTGAGGAGTGCTGTTTCCGCAGCTGTGACCTGGCCCTCCTGGAGACGTACTGTGCTACCCCCGCCAAGTCCGAGGGCGCGCCGgcacaccccggccgtcccagagcagtgcccacacagtgcgacgtcccccccaacagccgcttcgattgcgcccctgacaaggccatcacccaggaacagtgcgaggcccgcggctgctgctacatccctgcaaagcaggggctgcagggagcccagatggggcagccctggtgcttcttcccacccagctaccccagctacaagctggagaacctgagctcctctgaaatgggctacacggccaccctgacccgtaccacccccaccttcttccccaaggacatcctgaccctgcggctggacgtgatgatggagactgagaaccgcctccacttcacgatcaaagatccagctaacaggcgctacgaggtgccatggagaccccgcgtgtccacagccgggcaccgtccccactctacagcgtggagttctctgaggagcccttcggggtgatcgtgcaccggcagctggacggccgcgtgctgctgaacacgacggtggcgcccctgttattgcggaccagttccttcagctgtccacctcgctgccctcgcagtatatcacaggcctcgccgagcacctcagtmcctgatgctcagcaccagctggaccaggatcaccctgtggaaccgggaccttgcgcccacgcccggtgcgaacctctacgggtctcaccctttctacctggcgctggaggacggcgggtcggcacacggggtgttcctgctaaacagcaatgccatggatgtggtcctgcagccgagccctgccatagctggaggtcgacaggtgggatcctggatgtctacatcttcctgggcccagagcccaagagcgtggtgcagcagtacctggacgttgtgggatacccgttcatgccgccatactggggcctgggatccacctgtgccgctggggctactcctccaccgctatcacccgccaggtggtggagaacatgaccagggcccacttccccctggacgtccaatggaacgacctggactacatggactcccggagggacttcacgttcaacaaggatggatccgggacttcccggccatggtgcaggagctgcaccagggcggccggcgctacatgatgatcgtggatcctgccatcagcagctcgggccctgccgggagctacaggccctacgacgagggtctgcggaggggggttttcatcaccaacgagaccggccagccgctgattgggaaggtatggcccgggtccactgccttccccgacttcaccaaccccacagccctggcctggtgggaggacatggtggctgagaccatgaccaggtgccatcgacggcatgtggattgacatgaacgagccttccaacttcatcaggggctctgaggacggctgccccaacaatgagctggagaacccaccctacgtgcctggggtggttggggggaccctccaggcggcaaccatctgtgcctccagccaccagtttctctccacacactacaacctgcacaacctctacggcctgaccgaagccatcgcctcccacagggcgctggtgaaggctcgggggacacgcccatttgtgatctcccgctcgacctttgctggccacggccgatacgccggccactggacgggggacgtgtggagctcctgggagcagctcgcctcctccgtgccagaaatcctgcagtttaacctgctgggggtgcctctggtcggggccgacgtctgcggcttcctgggcaacacctcagaggagctgtgtgtgcgctggacccagctgggggccttctaccccttcatgcggaaccacaacagcctgctcagtctgccccaggagccgtacagcttcagcgagccggcccagcaggccatgaggaaggccctcaccctgcgctacgcactcctcccccacctctacacgctgttccaccaggcccacgtcgcgggggagaccgtggcccggcccctcttcctggagttccccaaggactctagcacctggactgtggaccaccagctcctgtggggggaggccctgctcatcaccccagtgctccaggccgggaaggccgaagtgactggctacttccccttgggcacatggtacgacctgcagacggtgccaatagaggcccttggcagcctcccacccccacctgcagctccccgtgagccagccatccacagcgaggggcagtgggtgacgctgccggcccccctggacaccatcaacgtccacctccgggctgggtacatcatccccctgcagggccaggcctcacaaccacagagtcccgccagcagcccatggccctggctgtggccctgaccaagggtggagaggcccgaggggagctgttctgggacgatggagagagcctggaagtgctggagcgaggggcctacacacaggtcatcttcctggccaggaataacacgatcgtgaatgagctggtacgtgtgaccagtgagggagctggcctgcagctgcagaaggtgactgtcctgggcgtggccacggcgccccagcaggtcctctccaacggtgtccctgtctccaacttcacctacagccccgacaccaaggtcctggacatctgtgtctcgctgttgatgggagagcagtttctcgtcagaggtgttagtctagagcttgctagcggccgcConstruct 1756

The GILTΔ2-7Δ34-40-GAA70-952 cassette below was cloned using the Asp718and NotI sites of the cassette and vector pCEP4 to producepCEP-GILTΔ2-7Δ34-40-GAA70-952 (Plasmid 1756). Restriction sites forcloning are in lowercase bold. The spacer amino acid sequence Gly, Ala,Pro (underlined sequence) separate the GAA gene and GILTΔ2-7Δ34-40 tag(upper case sequence). The spacer and tag are placed upstream of GAAresidue Ala70. The GILTΔ2-7Δ34-40 cassette contains a deletion of aminoacid residues 34-40 (Arg-Val-Ser-Arg-Arg-Ser-Arg) from the human IGF-IIsequence.

(SEQ ID NO: 30) ggtaccagctgctagcaagctaattcacaccaATGGGAATCCCAATGGGGAAGTCGATGCTGGTGCTTCTCACCTTCTTGGCCTTCGCCTCGTGCTGCATTGCTGCTCTGTGCGGCGGGGAGCTGGTGGACACCCTCCAGTTCGTCTGTGGGGACCGCGGCTTCTACTTCAGCAGGCCCGCAAGCGGCATCGTTGAGGAGTGCTGTTTCCGCAGCTGTGACCTGGCCCTCCTGGAGACGTACTGTGCTACCCCCGCCAAGTCCGAGGGCGCGCCGgcacaccccggccgtcccagagcagtgcccacacagtgcgacgtcccccccaacagccgcttcgattgcgcccctgacaaggccatcacccaggaacagtgcgaggcccgcggagctgctacatccctgcaaagcaggggctgcagggagcccagatggggcagccaggtgatatcccacccagctaccccagctacaagctggagaacctgagctcctctgaaatgggctacacggccaccctgacccgtaccacccccaccttcttccccaaggacatcctgaccctgcggctggacgtgatgatggagactgagaaccgcctccacttcacgatcaaagatccagctaacaggcgctacgaggtgccatggagaccccgcgtgtccacagccgggcaccgtccccactctacagcgtggagttactgaggagccatcggggtgatcgtgcaccggcagaggacggccgcgtgctgctgaacacgacggtggcgcccctgttctttgcggaccagttccttcagctgtccacctcgctgccctcgcagtatatcacaggcctcgccgagcacctcagtcccctgatgctcagcaccagctggaccaggatcaccctgtggaaccgggaccttgcgcccacgcccggtgcgaacctctacgggtctcaccctttctacctggcgctggaggacggcgggtcggcacacggggtgttcctgctaaacagcaatgccatggatgtggtcctgcagccgagccagcccttagctggaggtcgacaggtgggatcctggatgtctacatcttcctgggcccagagcccaagagcgtggtgcagcagtacctggacgttgtgggatacccgttcatgccgccatactggggcctgggcttccacctgtgccgctggggctactcctccaccgctatcacccgccaggtggtggagaacatgaccagggcccacttccccctggacgtccaatggaacgacctggactacatggactcccggagggacttcacgttcaacaaggatggcttccgggacttcccggccatggtgcaggagctgcaccagggcggccggcgctacatgatgatcgtggatcctgccatcagcagctcgggccctgccgggagctacaggccctacgacgagggtctgcggaggggggttttcatcaccaacgagaccggccagccgctgattgggaaggtatggcccgggtccactgcatccccgacttcaccaaccccacagccctggcctggtgggaggacatggtggctgagttccatgaccaggtgcccttcgacggcatgtggattgacatgaacgagccttccaacttcatcaggggctctgaggacggctgccccaacaatgagctggagaacccaccctacgtgcctggggtggttggggggaccctccaggcggcaaccatctgtgcctccagccaccagtttctctccacacactacaacctgcacaacctctacggcctgaccgaagccatcgcctcccacagggcgctggtgaaggctcgggggacacgcccatttgtgatctcccgctcgacctttgctggccacggccgatacgccggccactggacgggggacgtgtggagctcctgggagcagctcgcctcctccgtgccagaaatcctgcagtttaacctgctgggggtgcctaggtcggggccgacgtctgcggcttcctgggcaacacctcagaggagagtgtgtgcgctggacccagagggggccttctaccccttcatgcggaaccacaacagcctgctcagtagccccaggagccgtacagatcagcgagccggcccagcaggccatgaggaaggccacaccctgcgctacgcactcctcccccacctctacacgctgttccaccaggcccacgtcgcgggggagaccgtggcccggcccctatcctggagttccccaaggactctagcacctggactgtggaccaccagctcagtggggggaggccctgctcatcaccccagtgctccaggccgggaaggccgaagtgactggctacttcccatgggcacatggtacgacctgcagacggtgccaatagaggcccttggcagcctcccacccccacctgcagctccccgtgagccagccatccacagcgaggggcagtgggtgacgctgccggcccccctggacaccatcaacgtccacctccgggctgggtacatcatccccctgcagggccctggcctcacaaccacagagtcccgccagcagcccatggccaggctgtggccctgaccaagggtggagaggcccgaggggagagttctgggacgatggagagagcctggaagtgctggagcgaggggcctacacacaggtcatcttcctggccaggaataacacgatcgtgaatgagctggtacgtgtgaccagtgagggagctggcctgcagagcagaaggtgactgtcagggcgtggccacggcgccccagcaggtcctctccaacggtgtccctgtctccaacttcacctacagccccgacaccaaggtcctggacatctgtgtctcgagttgatgggagagcagtttctcgtcagctggtgttagtctagagcttgctagcggccgcConstruct 1763

The GILTΔ2-7M1/L27A37-GAA70-952 cassette below was cloned using theAsp718 and NotI sites of the cassette and vector pCEP4 to producepCEP-GILTΔ2-7M1/L27A37-GAA70-952 (Plasmid 1763). Restriction sites forcloning are in lowercase bold. The spacer amino acid sequence Gly, Ala,Pro (underlined sequence) separate the GAA gene and GILTΔ2-7M1/L27A37tag (upper case sequence). The spacer and tag are placed upstream of GAAresidue Ala70. The GILTΔ2-7M1/L27A37 cassette contains Y27L and R37Asubstitutions in the human IGFII sequence. The DNA sequence of the GILTcassette differs from the human DNA sequence at every 6^(th) codon.

(SEQ ID NO: 31) ggtaccaagcttgccATGGGAATCCCAATGGGCAAGTCGATGCTGGTGCTGCTCACCTTCTTGGCCTTTGCCTCGTGCTGCATTGCCGCTCTGTGCGGCGGGGAACTGGTGGACACCCTCCAATTCGTCTGTGGGGACCGGGGCTTCCTGTTCAGCAGACCCGCAAGCCGTGTGAGTGCTCGCAGCCGTGGCATTGTTGAGGAGTGCTGTTTTCGCAGCTGTGACCTGGCTCTCCTGGAGACGTACTGCGCTACCCCCGCCAAGTCTGAGGGCGCGCCGgcacaccccggccgtcccagagcagtgcccacacagtgcgacgtcccccccaacagccgcttcgattgcgcccctgacaaggccatcacccaggaacagtgcgaggcccgcggctgctgctacatccctgcaaagcaggggctgcagggagcccagatggggcagccaggtgcttatcccacccagctaccccagctacaagaggagaacctgagacctagaaatgggctacacggccaccagacccgtaccacccccaccttatccccaaggacatcctgaccctgcggaggacgtgatgatggagactgagaaccgcctccacttcacgatcaaagatccagctaacaggcgctacgaggtgcccttggagaccccgcgtgtccacagccgggcaccgtccccactctacagcgtggagttctctgaggagccatcggggtgatcgtgcaccggcagctggacggccgcgtgctgctgaacacgacggtggcgcccagttctttgcggaccagttccttcagagtccacctcgctgccctcgcagtatatcacaggcctcgccgagcacctcagtcccctgatgctcagcaccagaggaccaggatcaccagtggaaccgggaccttgcgcccacgcccggtgcgaacctctacgggtctcaccattctacctggcgctggaggacggcgggtcggcacacggggtgttcctgctaaacagcaatgccatggatgtggtcctgcagccgagccctgccatagaggaggtcgacaggtgggatcctggatgtctacatatcctgggcccagagcccaagagcgtggtgcagcagtacctggacgttgtgggatacccgttcatgccgccatactggggcagggcttccacctgtgccgctggggctactcaccaccgctatcacccgccaggtggtggagaacatgaccagggcccacttccccctggacgtccaatggaacgacctggactacatggactcccggagggacttcacgttcaacaaggatggcttccgggacttcccggccatggtgcaggagctgcaccagggcggccggcgctacatgatgatcgtggatcctgccatcagcagctcgggccctgccgggagctacaggccctacgacgagggtctgcggaggggggttttcatcaccaacgagaccggccagccgctgattgggaaggtatggcccgggtccactgccttccccgacttcaccaaccccacagccctggcctggtgggaggacatggtggctgagttccatgaccaggtgcccttcgacggcatgtggattgacatgaacgagccttccaacttcatcaggggctctgaggacggctgccccaacaatgagctggagaacccaccctacgtgcctggggtggttggggggaccctccaggcggcaaccatctgtgcctccagccaccagtttctctccacacactacaacctgcacaacctctacggcctgaccgaagccatcgcctcccacagggcgctggtgaaggctcgggggacacgcccatttgtgatctcccgctcgacctttgctggccacggccgatacgccggccactggacgggggacgtgtggagctcctgggagcagctcgcctcaccgtgccagaaatcctgcagtttaacctgctgggggtgcctctggtcggggccgacgtagcggcttcctgggcaacacctcagaggagctgtgtgtgcgaggacccagctgggggccttctaccccttcatgcggaaccacaacagcctgctcagtagccccaggagccgtacagcttcagcgagccggcccagcaggccatgaggaaggccctcaccctgcgctacgcactcctcccccacctctacacgctgttccaccaggcccacgtcgcgggggagaccgtggcccggcccacttcctggagttccccaaggactctagcacctggactgtggaccaccagctcctgtggggggaggccagctcatcaccccagtgctccaggccgggaaggccgaagtgactggctacttccccttgggcacatggtacgacctgcagacggtgccaatagaggcccttggcagcctcccacccccacctgcagctccccgtgagccagccatccacagcgaggggcagtgggtgacgctgccggcccccctggacaccatcaacgtccacctccgggctgggtacatcatccccagcagggccaggcctcacaaccacagagtcccgccagcagcccatggccaggctgtggccctgaccaagggtggagaggcccgaggggagctgttctgggacgatggagagagcctggaagtgctggagcgaggggcctacacacaggtcatcttcctggccaggaataacacgatcgtgaatgagaggtacgtgtgaccagtgagggagctggcctgcagagcagaaggtgactgtcctgggcgtggccacggcgccccagcaggtcctctccaacggtgtccctgtctccaacttcacctacagccccgacaccaaggtcctggacatctgtgtctcgctgttgatgggagagcagtttctcgtcagctggtgttagtctagagcttgctagcggccgc

Example 3 Expression and Purification of GILT-Tagged GAA Enzymes

Tissue Culture

GILT-tagged GAA plasmids were each transfected into suspension FreeStyle293-F cells as described by the manufacturer (Invitrogen). Briefly,cells were grown in Opti-MEM I media (Invitrogen) in polycarbonateshaker flasks on an orbital shaker at 37° C. and 8% CO₂. Cells wereadjusted to a concentration of 1×10⁶ cells/ml, then transfected with a1:1:1 ratio of ml DNA:μl 293fectin. Culture aliquots were harvested 5-7days post-transfection and centrifuged at 5,000×g for 5 minutes.Supernatants were stored frozen at −80° C.

Protein Purification and Concentration

Starting material was mammalian cell culture supernatant, as describedabove, thawed from storage at −80° C. Citric acid was added to reach pH6.0, then ammonium sulfate was added to reach a final concentration of1M. The material was passed through a 0.2 μm Supor-Mach filter(Nalgene).

The filtered material was loaded onto a Phenyl-Sepharose™ 6 Low-SubFast-Flow (GE Healthcare) column prepared with HIC Load Buffer (50 mMcitrate pH 6.0, 1M AmSO₄). The column was washed with 10 column volumesof HIC Wash Buffer (50 mM citrate pH 6.0, 0.8M AmSO₄), and eluted with 5column volumes of HIC Elution Buffer (50 mM citrate pH 6.0). Samplesfrom the elution peaks were pooled and buffer was exchanged intophosphate buffered saline (145.15 mM NaCl, 2.33 mM KCl, 10 mM Na₂HPO₄, 2mM KH₂PO₄, pH 6.2) using centricon spin concentrators (Amicon) andBio-Spin-6 de-salting columns (Bio-Rad).

Enzyme Activity

GAA expression was determined by a para-nitrophenol (PNP) enzymaticassay. GAA enzyme was incubated in 50 μl reaction mixture containing 100mM sodium acetate pH 4.2 and 10 mM Para-Nitrophenol (PNP) α-glucosidesubstrate (Sigma N1377). Reactions were incubated at 37° C. for 20minutes and stopped with 300 μl of 100 mM sodium carbonate. Absorbanceat 405 nm was measured in 96-well microtiter plates and compared tostandard curves derived from p-nitrophenol (Sigma N7660). 1 GAA PNP unitis defined as 1 nmole PNP hydrolyzed/hour.

Example 4 Competitive Receptor Binding Assays

The affinity of GILT-tagged proteins for the IGF2 receptor (IGF2R), IGF1receptor (IGF1R) and the insulin receptor (IR) was examined incompetitive binding experiments performed in a 96-well plate format.Receptors were coated at room temperature overnight onto Reacti-bindwhite plates (Pierce, Cat#437111) in Coating Buffer (0.05M Carbonatebuffer, pH 9.6) at a concentration of either 0.5 μg/well (IGF2R) or 1μg/well (IGF1R, IR). Plates were washed with wash buffer (PhosphateBuffered Saline plus 0.05% Tween-20), then blocked in Super BlockingBuffer (Pierce, Cat#37516) for 1 hour. After another plate washing,biotinylated ligands (Cell Sciences) were added to wells; IGF2R wellsreceived 8 nM IGF2-biotin, IGF1R wells received 30 nM IGF1-biotin, andIR wells received 20 nM insulin-biotin. Along with the biotinylatedligands, wells also contained serial dilutions of the GILT-tagged GAAprotein samples or non-biotinylated control ligands to act as bindinginhibitors for the biotinylated ligands. Following a two-hour rockingincubation, plates were washed and bound biotinylated ligands weredetected with a streptavidin-HRP incubation (R&D, Cat#890803, 1:200dilution in blocking buffer, 30 minutes), followed by a Super Elisa PicoChemiluminescent Substrate incubation (Pierce, Cat#37070, 5 minutes).The chemiluminescent signal was measured at 425 nm.

The percent bound biotinylated ligand was calculated for each competitorconcentration in the IGF2R binding competition assay and the IC₅₀ valueswere determined (FIG. 4). Protein 1752 with a deletion of IGF2 residues30-40 displayed a similar IC₅₀ value as the GILT-tagged ZC-701 (FIG. 4),indicating that deletion of these residues in the IGF2 loop region doesnot appear to effect IGF2R binding. Protein 1751 with a deletion of IGF2residues 29-40 displayed a higher IC₅₀ value (FIG. 4), indicating thatit does not compete as well for binding to the IGF2R.

On a separate IGF2R assay plate, comparison of ZC-701 and protein 1763yielded IC₅₀ values that differed by 35% (See FIG. 5).

In an assay measuring the competition of biotinylated insulin binding toplate-bound insulin, 1751 and 1752 proteins were not as effective asinhibitors compared to 701 or IGF-II (See FIG. 6). This indicates thatthe 1751 and 1752 proteins, with deletions in the loop regioncorresponding to amino acids 30-40 of the GILT tag, had a reducedaffinity for the insulin receptor compared to the intact GILT tag on 701or IGF-II.

In an assay measuring the competition of biotinylated IGF-I binding toplate-bound IGF1R, 1763 protein was not as effective as an inhibitorcompared to 701, IGF-II or IGF-I (See FIG. 7). This indicates that the1763 protein, with Δ2-7, Y27L and R37A mutations in the GILT tag, had areduced affinity for the IGF1 receptor compared to ZC-701 or IGF-II.

Example 4 Additional Insulin Receptor Binding Assay

Protein ZC-1487 was tested for its binding affinity for the insulinreceptor. Protein ZC-1487 contains the GILTD2-7M1/A37 cassette containswith and Arg to Ala substitution at amino acid 37 of the human IGF2sequence and is resistant to proteolysis by furin. Two different batchesof this protein purified from CHO cells, ZC-1487-B26 and ZC-1487-B28were analyzed in an assay measuring the competition of biotinylatedinsulin binding to plate-bound insulin.

An insulin receptor binding assay was conducted by competing insulin,IGF-II, ZC710B20 and ZC1487B26 or ZC-1487-B28 with Biotinylated-insulinbinding to the insulin receptor (Insulin-R).

Specifically, white Reacti-Bind™ plates were coated with Insulin-R at aconcentration of 1 ug/well/100 ul (38.4 nM). The coated plates wereincubate over night at room temperature, then washed 3× with washingbuffer (300 ul/well). The plates were then blocked with blocking buffer(300 ul/well) for 1 hour. The washing steps were repeated and any traceof solution in the plates was taken out.

Biotinylated-insulin was mixed at 20 nM with different concentrations ofinsulin, IGF-II, ZC701B20, B26 and B28 by serial dilutions (finalconcentrations are shown in Table 2). 100 ul of diluted Insulin, IGF-II,ZC710B20, ZC1487B26, and ZC1487B28 in 20 nM Insulin-biotin were addedinto the coated plates and the plates were incubated at room temperaturefor 2 hours. The plates were then washed 3 times with washing buffer.100 ul of strepavidin-HRP working solution (50 ul strepavidin-HRP in 10ml blocking buffer) was added into the plates and the plates wereincubated at room temperature for 30 minutes. 100 ul of Elisa-Picoworking solution containing Elisa-Pico chemiluminescent substrate wasadded and the chemiluminescence was measured at 425 nm. Exemplaryresults are shown in Table 2, FIG. 8, and FIG. 9. Both batches ofZC-1487 were not as effective as inhibitors compared to ZC-701 or theinsulin control. As can be seen from Table 2 and FIG. 8, furin resistantpeptide ZC-1487B26 binds to the insulin receptor more than 10-fold lessavidly than does ZC-701 and more than 20-fold less than does thewild-type IGF-II

This indicates that the 1487 protein had a reduced affinity for theinsulin receptor compared to the GILT tag on ZC-701.

TABLE 2 Insulin-Receptor Binding Activity - Chemiluminescence IntensityInsulin-B (nM) 2000 1000 500 250 125 62.5 31.25 15.625 7.8125 3.906251.95313 0 Insulin (nM) 20 nM 38.00 43.00 66.00 102.00 243.00 479.00750.00 780.00 503 1175 1046 2180 20 nM 13.00 25.00 57.00 141.00 229.00517.00 517.00 885.00 1003 1344 1462 1694 ave 25.5 34.0 61.5 121.5 236.0498.0 633.5 832.5 753.0 1259.5 1254.0 1937.0 IFG-II (nM) 20 nM 70.00268.00 356.00 644.00 828.00 991.00 1189.00 1492.00 1478 1478 1410 187420 nM 140.00 176.00 379.00 566.00 919.00 1224.00 1447.00 1377.00 14831370 1249 1959 ave 105.0 222.0 367.5 605.0 873.5 1107.5 1318.0 1434.51480.5 1424.0 1329.5 1916.5 Insulin-B (nM) 4000 2000 1000 500 250 12562.5 31.25 15.625 7.8125 3.90625 0 ZC701B20 (nM) 20 nM 191.00 387.00526.00 715.00 800.00 1284.00 1116.00 1248.00 1474 1241 1450 1790 20 nM250.00 329.00 483.00 774.00 767.00 1071.00 1024.00 968.00 1471 1118 12341886 ave 220.5 358.0 504.5 744.5 783.5 1177.5 1070.0 1108.0 1472.51179.5 1342.0 1838.0 ZC1487B26 (nM) 20 nM 967.00 1190.00 1334.00 1210.001294.00 1462.00 1402.00 1281.00 1323 1612 1173 1952 20 nM 962.00 1189.001395.00 1379.00 1612.00 1396.00 1221.00 1013.00 1326 1182 1102 2069 ave964.5 1189.5 1364.5 1294.5 1453.0 1429.0 1311.5 1147.0 1324.5 1397.01137.5 2010.5 2000 1000 500 250 125 62.5 31.25 5.625 7.8125 3.906251.95313 0 (4000) (2000) (1000) (500) (250) (125) (62.5) (31.25) (15.625)(7.8125) (3.90625)

Example 5 Uptake Assays

Some mutants were tested for retention of uptake activity. HEK293 cellswere transfected with constructs 1479 (R37K), 1487 (R37A) or ZC-701.After harvest, culture supernatants were partially purified by HICchromatography. All samples were treated with PNGase prior toelectrophoresis.

FIG. 10 shows partially purified preparations of targeted fusionproteins containing a furin-resistant IGF-II mutein tag analyzed bySDS-PAGE and immunoblotting. As can be seen, the fusion protein encodedby construct 1487 containing R37A mutation is resistant to exogenousfurin.

FIG. 11 illustrates exemplary uptake results of furin resistantGILT-tagged GAA into rat L6 myoblasts. As shown in FIG. 11, exemplaryK_(uptakes) for proteins 1479, 1487, ZC-701, and purified ZC-701 are 4.5nM, 4.4 nM, 5.0 nM and 2.6 nM, respectively, which indicates that theproteins encoded by constructs 1487 (R37A) and 1479 (R37K) retain theability for efficient uptake into rat L6 myoblasts. The efficient uptakeof fusion proteins containing a furin-resistant GILT tag also indicatesthat the furin-resistant tag retains high affinity for the CI-MPR.

EQUIVALENTS

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments of the invention described herein. The scope of the presentinvention is not intended to be limited to the above Description, butrather is as set forth in the appended claims. The articles “a”, “an”,and “the” as used herein in the specification and in the claims, unlessclearly indicated to the contrary, should be understood to include theplural referents. Claims or descriptions that include “or” between oneor more members of a group are considered satisfied if one, more thanone, or all of the group members are present in, employed in, orotherwise relevant to a given product or process unless indicated to thecontrary or otherwise evident from the context. The invention includesembodiments in which exactly one member of the group is present in,employed in, or otherwise relevant to a given product or process. Theinvention also includes embodiments in which more than one, or all ofthe group members are present in, employed in, or otherwise relevant toa given product or process. Furthermore, it is to be understood that theinvention encompasses variations, combinations, and permutations inwhich one or more limitations, elements, clauses, descriptive terms,etc., from one or more of the claims is introduced into another claimdependent on the same base claim (or, as relevant, any other claim)unless otherwise indicated or unless it would be evident to one ofordinary skill in the art that a contradiction or inconsistency wouldarise. Where elements are presented as lists, e.g., in Markush group orsimilar format, it is to be understood that each subgroup of theelements is also disclosed, and any element(s) can be removed from thegroup. It should it be understood that, in general, where the invention,or aspects of the invention, is/are referred to as comprising particularelements, features, etc., certain embodiments of the invention oraspects of the invention consist, or consist essentially of, suchelements, features, etc. For purposes of simplicity those embodimentshave not in every case been specifically set forth herein. It shouldalso be understood that any embodiment of the invention, e.g., anyembodiment found within the prior art, can be explicitly excluded fromthe claims, regardless of whether the specific exclusion is recited inthe specification.

It should also be understood that, unless clearly indicated to thecontrary, in any methods claimed herein that include more than one act,the order of the acts of the method is not necessarily limited to theorder in which the acts of the method are recited, but the inventionincludes embodiments in which the order is so limited. Furthermore,where the claims recite a composition, the invention encompasses methodsof using the composition and methods of making the composition. Wherethe claims recite a composition, it should be understood that theinvention encompasses methods of using the composition and methods ofmaking the composition.

INCORPORATION OF REFERENCES

All publications and patent documents cited in this application areincorporated by reference in their entirety to the same extent as if thecontents of each individual publication or patent document wereincorporated herein.

What is claimed is:
 1. A targeted therapeutic fusion protein comprising:a lysosomal enzyme which is α-N-Acetylglucosaminidase (Naglu); an IGF-IImutein comprising amino acids 8-67 of SEQ ID NO: 1 and an Alasubstitution at position Arg37 of SEQ ID NO:1, wherein the IGF-II mutein(i) has diminished binding affinity for the insulin receptor relative tothe affinity of naturally-occurring human IGF-II for the insulinreceptor, (ii) is resistant to furin cleavage and (iii) binds to thehuman cation-independent mannose-6-phosphate receptor in amannose-6-phosphate-independent manner; and a spacer between thelysosomal enzyme and the IGF-II mutein, wherein the spacer comprises theamino acid sequence Gly-Ala-Pro.
 2. A pharmaceutical compositionsuitable for treating lysosomal storage disease comprising a targetedtherapeutic fusion protein of claim 1 and a physiologically acceptablecarrier or excipient.
 3. The targeted therapeutic fusion protein ofclaim 1, wherein the IGF-II mutein is fused via the spacer to theC-terminus of the lysosomal enzyme.
 4. The targeted therapeutic fusionprotein of claim 1, wherein the IGF-II mutein is fused via the spacer tothe N-terminus of the lysosomal enzyme.
 5. The targeted therapeuticfusion protein of claim 1, wherein the IGF-II mutein consists of aminoacids 8-67 of SEQ ID NO:1 having an Ala substitution at position Arg37of SEQ ID NO:1.